Carbohydrate-enriched recombinant microorganisms

ABSTRACT

The present disclosure relates to recombinant microorganisms engineered for enhanced production of a desired carbohydrate, as well as related biomass, and compositions which are useful, inter alia, as animal feed ingredients. The present disclosure also provides related methods.

STATEMENT REGARDING SEQUENCE LISTING

The “Sequence Listing” submitted electronically concurrently herewithpursuant 37 C.F.R. § 1.821 in computer readable form (CRF) via EFS-Webas file name 200206_416D1_SEQUENCE LISTING.txt is incorporated herein byreference. The electronic copy of the Sequence Listing was created onMay 10, 2019, and the size is 277 KB.

TECHNICAL FIELD

The present disclosure relates to novel recombinant C₁ metabolizingmicroorganisms comprising an engineered metabolic pathway for theenhanced production of carbohydrates, and related compositions andmethods.

BACKGROUND

Advances in the efficiency in animal feed utilization have been achievedover the past several decades through the use of feed additives. Theseadded substances augment the nutrient-content, energy-content, and/ordisease fighting properties of animal feed compositions. A growingchallenge for commercial animal producers is the rising cost of grain.The rising costs are due in part to competing demands for grains forbiofuel and human food use. With the rising cost of grain and proteincomponents, coupled with limited land available for feed production,alternative low cost animal feed products with beneficial nutritive anddisease fighting properties would be highly desirable.

SUMMARY

In one embodiment, the present disclosure provides a recombinant C₁metabolizing microorganism comprising an exogenous nucleic acid selectedfrom the group consisting of an exogenous nucleic acid that encodes acarbohydrate biosynthesis enzyme and an exogenous nucleic acid thatencodes an expression control sequence that is operably linked to anucleic acid encoding a native carbohydrate biosynthesis enzyme, whereinthe recombinant C₁ metabolizing microorganism is capable of converting anatural gas-derived carbon feedstock into a desired carbohydrate.Typically, the natural gas-derived carbon feedstock is natural gas ormethane.

In another embodiment, the present disclosure provides a biomass derivedfrom the recombinant C₁ metabolizing microorganism of the presentdisclosure.

In a further embodiment, the present disclosure provides a carbohydratecomposition comprising carbohydrates extracted from the biomass of thepresent disclosure, wherein the composition exhibits a δ¹³C that is lessthan −30‰.

In a still further embodiment, the present disclosure provides an animalfeed comprising the biomass of the present disclosure.

In another embodiment, the present disclosure provides a culture orfermentation medium comprising the biomass or composition of the presentdisclosure.

The present disclosure additionally provides related methods.

DETAILED DESCRIPTION

The instant disclosure provides novel recombinant C₁ metabolizingmicroorganisms that have the ability to utilize relatively low-costcarbon feedstock as an energy source, as well as related biomass,compositions, and methods. The recombinant microorganisms of the presentdisclosure are engineered for the enhanced production of certaincarbohydrates that are commercially desirable. These recombinantmicroorganisms, as well as the biomass and carbohydrate compositionsthat are derived from them, are useful as a source of nutrition foranimals (such as, for example, livestock, fish, poultry, and the like),as well as cultured or fermented microorganisms.

In one embodiment, the present disclosure provides a recombinant C₁metabolizing microorganism, wherein the recombinant C₁ metabolizingmicroorganism comprises an exogenous nucleic acid selected from thegroup consisting of an exogenous nucleic acid that encodes acarbohydrate biosynthesis enzyme and an exogenous nucleic acid thatencodes an expression control sequence that is operably linked to anucleic acid encoding a native carbohydrate biosynthesis enzyme, whereinthe recombinant C₁ metabolizing microorganism is capable of converting anatural gas carbon feedstock into the carbohydrate. When theserecombinant microorganisms are cultured in the presence of a naturalgas-derived C₁ substrate, they typically exhibit a δ¹³C of less than−30‰, and often less than −40‰, as described in more detail herein.Typically, the recombinant microorganism is a non-photosynthetic C₁metabolizing microorganism.

In these embodiments, the recombinant microorganisms of the presentdisclosure are engineered to convert a natural gas-derived feedstock,which is a relatively low cost and abundant resource (for example,natural gas, or a C₁ substrate such as methane from natural gas) ascompared to more costly carbohydrates, to higher valued carbohydrates.As used herein, the term “natural gas-derived feedstock” refers tonatural gas, or any of the components isolated from natural gas(including C₁ substrates) or converted from natural gas (i.e., syngas).

The term “natural gas” refers herein to naturally occurring gas mixturesthat may be obtained by conventional processes (e.g., drilling and waterflooding of porous reservoirs) or non-conventional processes (e.g.,hydraulic fracturing, horizontal drilling or directional drilling offormations having low gas permeability). The gas mixtures are made up ofmethane and other compounds, including other C₁ compounds, as well asother light alkane gases (such as, for example, ethane, propane, butane,pentane, and the like), carbon dioxide, nitrogen, hydrogen sulfide, orthe like, and combinations thereof. Unconventional natural gas may beobtained from sources such as, for example, tight gas sands formed insandstone or carbonate, coal bed methane formed in coal deposits andadsorbed in coal particles, shale gas formed in fine-grained shale rockand adsorbed in clay particles or held within small pores ormicrofractures, methane hydrates that are a crystalline combination ofnatural gas and water formed at low temperature and high pressure inplaces such as under oceans and permafrost.

As used herein, “C₁ substrate” or “C₁ compound” refers to any carboncontaining molecule or composition that lacks a carbon-carbon bond.Exemplary C₁ substrates include syngas, methane, methanol, formaldehyde,formic acid or a salt thereof, carbon monoxide, carbon dioxide,methylated amines (e.g., methylamine, dimethylamine, trimethylamine,etc.), methylated thiols, methyl halogens (e.g., bromomethane,chloromethane, iodomethane, dichloromethane, etc.), cyanide, or anycombination thereof.

In certain embodiments of the present disclosure, a natural gas-derivedfeedstock may be natural gas, a C₁ substrate from natural gas, orsyngas. Typically, a C₁ substrate is methane. Exemplary recombinant C₁metabolizing microorganisms that have utilized a natural gas-derivedcarbon substrate as a feedstock exhibit a distinctive isotopic carbonsignature, which is described in more detail herein. This distinctiveisoptopic carbon signature is also exhibited by the compositions andproducts of such recombinant microorganisms (e.g., biomass, carbohydratecompositions, and the like).

In another embodiment, the present disclosure provides a recombinant C₁metabolizing microorganism comprising an exogenous nucleic acid encodinga carbohydrate biosynthesis enzyme, wherein the C₁ metabolizingmicroorganism is capable of converting methane into a carbohydrate.Exemplary carbohydrates are glucans. In some embodiments, a carbohydrateis a β-(1,3)-glucan, and may be branched or unbranched or a mixturethereof. Usually, a C₁ metabolizing microorganism is anon-photosynthetic C₁ metabolizing microorganism.

As used herein, “C₁ metabolizing microorganism” or “C₁ metabolizingnon-photosynthetic microorganism” refers to any microorganism having theability to use a C₁ substrate as a source of energy or as its primarysource of energy and biomass, and may or may not use other carbonsubstrates (such as sugars and complex carbohydrates) for energy andbiomass. For example, a C₁ metabolizing microorganism may oxidize a C₁substrate, such as methane or methanol. C₁ metabolizing microorganismsinclude bacteria (such as methanotrophs and methylotrophs) and yeast. Incertain embodiments, a C₁ metabolizing microorganism does not include aphotosynthetic microorganism, such as algae. In some embodiments, the C₁metabolizing microorganism will be an “obligate C₁ metabolizingmicroorganism,” meaning its sole source of energy are C₁ substrates. Infurther embodiments, a C₁ metabolizing microorganism (e.g.,methanotroph) will be cultured in the presence of a C₁ substratefeedstock (i.e., using the C₁ substrate as a source of energy).

Recombinant C₁ metabolizing microorganisms of the present disclosure areengineered for enhanced production of a desired carbohydrate and in oneembodiment, comprise an exogenous nucleic acid encoding a carbohydratebiosynthesis (CB) enzyme. The terms “carbohydrate biosynthesis enzyme”and “CB enzyme” are used interchangeably herein to refer to an enzymethat is involved in the production of a carbohydrate by the recombinanthost C₁ metabolizing microorganism.

Exogenous nucleic acids encoding CB enzymes that are employed in thepractice of the present disclosure are typically codon optimized foroptimal expression from the recombinant host C₁ metabolizingmicroorganism and encode an enzyme that is either native to a speciesheterologous to the host C₁ microorganism or is a mutant (i.e., variant)of an enzyme that exists in nature.

As used herein, the term “carbohydrate” refers to a monosaccharide, adisaccharide, or a polysaccharide. Suitable exogenous nucleic acidsemployed in the practice of the present disclosure include those whichencode enzymes that are involved in the production of a monosaccharidesuch as, for example, glucose, fructose, ribose, glyceraldehyde,galactose and the like; a disaccharide, such as, for example lactose,sucrose, maltose, cellulobiose, and the like, and mixtures thereof or apolysaccharide, including, for example, an unbranched or branchedglucan, and the like, and mixtures thereof. Exemplary glucans includeα-glucans, such as for example, dextran, glycogen, pullulan, starch, andthe like, as well as β-glucans, such as, for example, β-1,4-glucan(i.e., cellulose), β-1,3-glucan, β-(1,3)(1,4)-glucan,β-(1,3)(1,6)-glucan, and the like, and mixtures thereof.

In a specific embodiments, the CB enzyme is an enzyme involved in theproduction of an unbranched or a branched glucan, or mixture thereofβ-glucans are known to have beneficial therapeutic properties, includingas a powerful immune stimulant and a powerful antagonist to both benignand malignant tumors. β-glucans are also known to lower cholesterol andtriglyceride levels. See D. Akramiené et al., Medicina (kaunas), 2007;43(8):597. The β-glucans are a heterogeneous group of glucose polymersmade up of β-D-glucopyranosyl units having β-(1,3) and/or β-(1,4),and/or β-(1,6) linkages. They have been isolated from a number ofsources, including plants (oat, barley, bran, seaweed, corn, soy, andthe like), bacteria (e.g., Pneumocystis carinii, Cryptococcusneoformans, Aspergillus fumigatus, Histoplasma capsulatum, Candidaalbicans, and the like), and fungi (i.e., Saccharomyces cerevisiae andmushrooms, such as, for example shiitake (Lentinus edodes), maitake(Grifola frondosa), schizophylan (Schizophillum commune), and SSG(Sclerotinia sclerotiorum). β-glucan extracts from Lentinus edodes andSchizophillum commune have been used for the treatment of cancer inJapan since 1980. Id.

Exogenous nucleic acids that are suitable for use in the practice of thepresent disclosure include those which encode enzymes involved ingluconeogenesis, glycogenesis, α- or β-glucan biosynthesis, and othermetabolic pathways known to produce a carbohydrate.

Suitable exogenous nucleic acids include those which encode agluconeogenesis enzyme selected from the group consisting of a pyruvatecarboxylase, a phosphoenolpyruvate carboxykinase, an enolase, aphosphoglycerate mutase, a phosphoglycerate kinase, aglyceraldehyde-3-phosphate dehydrogenase, a Type A aldolase, a fructose1,6-bisphosphatase, a phosphofructokinase, a phosphoglucose isomerase, ahexokinase, a glucose-6-phosphate, and the like.

Other suitable exogenous nucleic acids include those which encode aglycogenesis enzyme selected from the group consisting of aglucose-1-phosphate adenyltransferase, a glycogen synthase, and thelike.

The above enzymes can be found in a number of heterologous species,including microorganisms, such as, for example, bacteria and yeast,including, for example, E. coli, C. glutamicum, Saccharomycescerevisiae, and the like, as well as higher order fungi, such asmushrooms, and the like, as well as algae, and plants.

Suitable exogenous nucleic acids include those which encode a glucanbiosynthesis enzyme, such as, for example, a glucan synthase. Anexemplary glucan synthase is β-1,3-glucan synthase. The exogenousnucleic acid may encode a glucan biosynthesis enzyme (e.g., a glucansynthase (such as, for example a β-1,3-glucan synthase)) from a plant(oat, barley, bran, seaweed, corn, soy, and the like), a bacteria (e.g.,Pneumocystis carinii, Cryptococcus neoformans, Aspergillus fumigatus,Histoplasma capsulatum, Candida albicans, and the like), or a fungi(i.e., Saccharomyces cerevisiae and mushrooms, such as, for exampleshiitake (Lentinus edodes), maitake (Grifola frondosa), schizophylan(Schizophillum commune), and SSG (Sclerotinia sclerotiorum). The aminoacid and nucleic acid sequences of a number of β-(1,3)-glucan synthasesare known. See, e.g., U.S. Pat. No. 5,194,600, WO99/49047, and EP 0 724644 B1, all of which are incorporated herein by reference. In certainspecific embodiments, the exogenous nucleic acid encodes a carbohydratebiosynthesis enzyme having the amino acid sequence of any of SEQ NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38,shown in Table A, hereinbelow. As described above, the exogenous nucleicacid is typically codon optimized for optimal expression from therecombinant C₁ microorganism. Exemplary nucleic acid sequences encodingthese CB enzymes are also provided in Table A. These nucleic acidsequences have been codon optimized for expression in Methylococcuscapsulatus Bath.

TABLE A Exemplary Carbohydrate Biosynthesis Enzymes Amino Acid NucleicAcid Sequence Sequence Source/Enzyme Name (SEQ ID NO.) (SEQ ID NO.)Saccharomyces cerevisiae: 2 1 mature KRE1 protein Saccharomycescerevisiae: 4 3 mature KRE2 protein Saccharomyces cerevisiae 6 5 s288c:FKS1 Saccharomyces cerevisiae: 8 7 FKS2 Candida albicans: FKS1 10 9 Zeamays (corn): portion of 12 11 1,3-β-D-glucan synthase Zea mays (corn):portion of 14 13 1,3-β-D-glucan synthase Oryza sativa (rice): portion of16 15 1,3-beta-D-glucan synthase Oryza sativa (rice): portion of 18 171,3-beta-D-glucan synthase Gycine max (soy): portion of 20 191,3-beta-D-glucan synthase. Veronia mespilifolia: 22 211,3-beta-D-glucan synthase Triticum aestivum (wheat): 24 231,3-beta-D-glucan synthase Horderum vulgars (barley): 26 251,3-beta-D-glucan synthase E. coli: Glucose-1-phosphate 28 27adenyltransfersase (Acc. No. YP 49003.1) Cornebacterium. Glutamicum 3029 (ATCC 13032): Glucose-1- phosphate adenylyltransferase Escherichiacoli str. K-12 32 31 substr. W3110: Glycogen Synthase Cornebacteriumglutamicum 34 33 (ATCC 13032): Glycosyltransferase E. coli:1,4-alpha-glucan 36 35 branching enzyme (Acc. No. YP 492001.1)Corynebacterium glutamicum 38 37 (ATCC 13032): Glycogen branching enzyme

Suitable exogenous nucleic acids employed in the practice of the presentdisclosure include those which encode a variant CB enzyme sequence thatis at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a reference or parental wild-type polypeptide sequence,such as, for example a reference sequence corresponding to any one ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, or 38, provided that the variant retains the carbohydratebiosynthesis enzyme activity of interest. In certain embodiments, the CBenzyme variant polypeptides will include at least one amino acidsubstitution (e.g., 1, 2, 3, 5, 6, 7, 8, 9 or 10 or more or up to 20,25, or 30 substitutions) at a pre-determined position relative to areference or parental wild-type CB enzyme, provided that a variantretains the CB enzyme activity of interest. The CB enzyme variantpolypeptides may further comprise one or more conservativesubstitutions. A “conservative substitution” is recognized in the art asa substitution of one amino acid for another amino acid that has similarproperties. Exemplary conservative substitutions are well known in theart (see, e.g., WO 97/09433, p. 10; Lehninger, Biochemistry, 2^(nd)Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-′7′7; Lewin, GenesIV, Oxford University Press, NY and Cell Press, Cambridge, Mass. (1990),p. 8, which are incorporated herein by reference). Methods forgenerating suitable exogenous nucleic acids encoding such variantenzymes are described in more detail herein.

The “percent identity” between two or more nucleic acid or amino acidsequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical positions/totalnumber of positions×100), taking into account the number of gaps, andthe length of each gap that needs to be introduced to optimize alignmentof two or more sequences. The comparison of sequences and determinationof percent identity between two or more sequences can be accomplishedusing a mathematical algorithm, such as BLAST and Gapped BLAST programsat their default parameters (e.g., Altschul et al., J. Mol. Biol.215:403, 1990; see also BLASTN at the world wide web atncbi.nlm.nih.gov/BLAST, which are incorporated herein by reference).

As indicated above, the exogenous nucleic acids encoding CB enzymesemployed in the practice of the present disclosure may be codonoptimized for expression in the C₁ metabolizing microorganism.Expression of recombinant proteins may be difficult outside theiroriginal host. For example, variation in codon usage bias has beenobserved across different species of bacteria (Sharp et al., Nucl.Acids. Res. 33:1141, 2005, which is incorporated herein by reference).Overexpression of recombinant proteins even within their native host mayalso be difficult. In certain embodiments, the nucleic acid to beintroduced into a host as described herein may be subjected to codonoptimization prior to introduction into the host to ensure proteinexpression is effective or enhanced. Codon optimization refers toalteration of codons in genes or coding regions of nucleic acids beforetransformation to reflect the typical codon usage of the host withoutaltering the polypeptide encoded by the non-natural DNA molecule. Codonoptimization methods for optimum gene expression in heterologous hostshave been previously described (see, e.g., Welch et al., PLoS One4:e7002, 2009; Gustafsson et al., Trends Biotechnol. 22:346, 2004; Wu etal., Nucl. Acids Res. 35:D76, 2007; Villalobos et al., BMCBioinformatics 7:285, 2006; U.S. Patent Publication Nos. 2011/0111413and 2008/0292918; disclosure of which methods are incorporated herein byreference, in their entirety). Exogenous nucleic acids encoding CBenzymes that are suitable for use in the practice of the presentdisclosure include those having a nucleic acid sequence that is at leastabout 85% identical to a nucleic acid reference sequence selected fromthe group consisting of SEQ ID NO.:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, theexogenous nucleic acid encoding the CB enzyme has a nucleic acidsequence that is at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98% and at least about 99% sequence identity to a nucleicacid reference sequence selected from the group consisting of SEQ IDNO.:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,and 37. Illustrative exogenous nucleic acids that encode a CB enzymewhich are suitable for use in the practice of the invention includesequences which have been codon optimized for optimal expression inMethylococcus capsulatus Bath, such as, for example, any one of SEQ IDNO.:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,and 37.

Similarly, exogenous nucleic acid molecules of this disclosure encodingpolypeptide variants may be designed using the phylogenetic-basedmethods described in the references noted above (U.S. Pat. No.8,005,620; Gustafsson et al.; Welch et al.; Villalobos et al.; Minshullet al., all of which are incorporated herein by reference.).

An exogenous nucleic acid encoding a carbohydrate biosynthesis enzymeincludes polynucleotides that encode a polypeptide, a polypeptidefragment, a peptide, or a fusion polypeptide that has or retains thecorresponding carbohydrate biosynthesis enzyme activity. Methods todetermine whether a polypeptide has a particular activity by measuringthe ability of the polypeptide to convert a substrate into a product areknown in the art.

In some embodiments, the exogenous nucleic acid encodes an expressioncontrol sequence that is operably linked to a nucleic acid encoding anative carbohydrate biosynthesis enzyme. Typically, the expressioncontrol sequence is one that results in the overexpression of a nativecarbohydrate biosynthesis enzyme. As used herein, “overexpressed” and“overexpression” when referring to a gene or a protein means an increasein expression or activity of the gene or protein. Increased expressionor activity includes expression or activity of a gene or protein beingincreased above the level of a wildtype (native or non-geneticallyengineered) control or reference microorganism. A gene or protein isoverexpressed if the expression or activity is in a microorganism whereit is not normally expressed or active. A gene or protein isoverexpressed if the expression or activity is extended or presentlonger in the recombinant microorganism than in a wild-type control orreference microorganism.

In addition to the exogenous nucleic acids described hereinabove,recombinant C₁ metabolizing microorganisms of the present disclosure maycomprise further genetic modifications which enhance the production ofthe desired carbohydrate. For example, when the exogenous nucleic acidencodes a carbohydrate biosynthesis enzyme, the recombinant C₁metabolizing microorganism may further comprise an exogenous expressioncontrol sequence that is operatively linked to the exogenous nucleicacid encoding the carbohydrate biosynthesis enzyme to enhance productionof the desired carbohydrate. Expression control sequences suitable foruse in the practice of the present disclosure are described in moredetail herein.

Alternatively, or in addition, the recombinant C₁ metabolizingmicroorganism of the present disclosure may further comprise anexogenous expression control sequence operatively linked to anendogenous nucleic acid encoding an endogenous enzyme that utilizes oneor more of the same substrates utilized by carbohydrate biosynthesisenzymes, or utilizes the desired carbohydrate as a substrate (i.e., a“competing” endogenous enzyme). This may be done to downregulate thecompeting endogenous enzyme.

In some embodiments, it may be desirable to reduce or inhibit acompeting endogenous enzyme activity by mutating the competingendogenous enzyme to delete or attenuate its activity. “Inhibit” or“inhibited,” as used herein, refers to an alteration, reduction, downregulation, abrogation or deletion, directly or indirectly, in theexpression of a target gene or in the activity of a target moleculerelative to a control, endogenous or reference molecule, wherein thealteration, reduction, down regulation or abrogation is statistically,biologically, industrially, or clinically significant.

Various methods for downregulating, inactivating, knocking-out, ordeleting endogenous gene function in C₁ metabolizing microorganisms areknown in the art. For example, targeted gene disruption is an effectivemethod for gene down-regulation where an exogenous polynucleotide isinserted into a structural gene to disrupt transcription. Geneticcassettes comprising the exogenous insertion DNA (e.g., a geneticmarker) flanked by sequence having a high degree of homology to aportion of the target host gene to be disrupted are introduced into thehost C₁ metabolizing microorganism. Exogenous DNA disrupts the targethost gene via native DNA replication mechanisms. Allelic exchange toconstruct deletion/insertional mutants in C₁ metabolizingmicroorganisms, including methanotrophic bacteria, have been describedin, for example, Toyama and Lidstrom, Microbiol. 144:183, 1998; Stoylaret al., Microbiol. 145:1235, 1999; Ali et al., Microbiol. 152:2931,2006; Van Dien et al., Microbiol. 149:601, 2003; Martin and Murrell,FEMS Microbiol. Lett. 127:243, 2006, all of which are incorporatedherein by reference.

For example, in some embodiments of the present disclosure, arecombinant C₁ metabolizing microorganisms may further comprise adeletion of endogenous glycogen synthase activity and/or endogenousphosphoglucomutase activity. Enzymes involved in other pathways, such asan amino acid synthesis pathway, may also be targeted for downregulation to focus metabolic activities of the host microorganism oncarbohydrate biosynthesis.

The recombinant C₁ metabolizing microorganism may thus be engineered tohave the ability to produce the desired carbohydrate at enhanced levels.In some of these embodiments, a recombinant C₁ metabolizingmicroorganism produces the desired carbohydrate at a level that is atleast about 10% greater than that produced by the native C₁ metabolizingmicroorganism and up to about 2-fold, to about 3-, 4-, 5-, 10-, 20-,30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, and up to about 500- or about1000-fold the level produced by a native C₁ metabolizing microorganism,when cultured in the presence of a natural gas-derived feedstock (e.g.,natural gas, methane, and the like) under at least one set of cultureconditions. In other embodiments, a recombinant C₁ metabolizingmicroorganism produces the desired carbohydrate at a level that is fromat least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or is at least about 95% greaterthan that produced by a native C₁ metabolizing microorganism, and up toabout 2-fold, to about 3-, 4-, 5-, 10-, 20-, 30-, 40-, 50-, 60-, 70-,80-, 90-, 100-, to about 500- or about 1000-fold the level produced bythe native C₁ metabolizing microorganism, when cultured in the presenceof a natural gas-derived feedstock under at least one set of cultureconditions. Typically, the enhanced level of production of a desiredcarbohydrate by a recombinant C₁ metabolizing microorganism of thepresent invention is at least about 2-fold, 3-, 4-, 5-, 10-, 20-, 30-,40-, 50-, 60-, 70-, 80-, 90-, or 100-fold that of the native C₁metabolizing microorganism, when cultured in the presence of a naturalgas-derived feedstock under at least one set of culture conditions.

Recombinant methods for expression of exogenous nucleic acids inmicrobial organisms are well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999), all of which are incorporatedherein by reference. Genetic modifications to nucleic acid moleculesencoding enzymes, or functional fragments thereof, can confer abiochemical or metabolic capability to a recombinant cell that isaltered from its naturally occurring state.

As used herein, the terms “endogenous” and “native” when referring to anucleic acid, polypeptide, such as an enzyme, compound or activityrefers to a nucleic acid, polypeptide, compound or activity that isnormally present in a host cell. The term “homologous” or “homolog”refers to a molecule or activity from an exogenous (non-native) sourcethat is the same or similar molecule or activity as that found in orderived from a host cell, species or strain.

As used herein, the term “exogenous” when referring to a nucleic acidmolecule, construct or sequence refers to a nucleic acid molecule orportion of a nucleic acid molecule sequence that is not native to a cellin which it is expressed, a nucleic acid molecule or portion of anucleic acid molecule native to a host cell that has been altered ormutated, or a nucleic acid molecule with an altered expression ascompared to the native expression levels under similar conditions. Forexample, an exogenous control sequence (e.g., promoter, enhancer) may beused to regulate expression of a gene or a nucleic acid molecule in away that is different than the gene or a nucleic acid molecule that isnormally expressed in nature or culture. In certain embodiments, anexogenous nucleic acid molecule may be homologous to a native host cellgene, but may have an altered expression level or have a differentsequence or both. In other embodiments, exogenous nucleic acid moleculesmay not be endogenous to a host cell or host genome, but instead mayhave been added to a host cell by conjugation, transformation,transfection, electroporation, or the like, wherein the added moleculemay integrate into the host genome or can exist as extra-chromosomalgenetic material (e.g., plasmid or other self-replicating vector).

In certain embodiments, more than one exogenous nucleic acid moleculecan be introduced into a host cell as separate nucleic acid molecules,as a polycistronic nucleic acid molecule, as a single nucleic acidmolecule encoding a fusion protein, or any combination thereof, andstill be considered as more than one exogenous nucleic acid. Forexample, a C₁ metabolizing microorganism can be modified to express twoor more exogenous nucleic acid molecules, which may be the same ordifferent, that encode one or more carbohydrate biosynthesis enzyme asdisclosed herein. In certain embodiments, multiple copies of acarbohydrate biosynthesis enzyme-encoding polynucleotide molecule areintroduced into a host cell, which may be two, three, four, five, six,seven, eight, nine, ten or more copies of the same carbohydratebiosynthesis enzyme or different carbohydrate biosynthesis enzymeencoding polynucleotides.

Host Cells and Transformation Methods

In carrying out the practice of the present invention, the exogenousnucleic acids described hereinabove are transformed into a host cellthat is a C₁ metabolizing microorganism. The C₁ metabolizingmicroorganism employed may be natural, strain adapted (e.g., performingfermentation to select for strains with improved growth rates andincreased total biomass yield compared to the parent strain), orrecombinantly modified to produce or overexpress the carbohydratebiosynthesis enzyme of interest and/or to have increased growth rates.Typically, the C₁ metabolizing microorganism is a non-photosynthetic C₁microorganism (e.g., is not an algae or a plant).

In certain embodiments, the present disclosure employs C₁ metabolizingmicroorganisms that are prokaryotes or bacteria, such as Methylomonas,Methylobacter, Methylococcus, Methylosinus, Methylocystis,Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus,Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus,Nocardia, Arthrobacter, Rhodopseudomonas, or Pseudomonas.

In further embodiments, the C₁ metabolizing bacteria employed is amethanotroph or a methylotroph. Exemplary methanotrophs includeMethylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis,Methylomicrobium, Methanomonas, Methylocella, or a combination thereof.Exemplary methylotrophs include Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium populi,Methylobacterium chloromethanicum, Methylobacterium nodulans, or acombination thereof. As used herein, the term “methylotrophic bacteria”refers to any bacteria capable of oxidizing any compound in any form(e.g., solid, liquid, gas) that contains at least one carbon and that donot contain carbon-carbon bonds. In certain embodiments, amethylotrophic bacterium may be a methanotroph. For example,“methanotrophic bacteria” refers to any methylotrophic bacteria thathave the ability to oxidize methane as a source of carbon and energy,which may be the primary source of carbon and energy. Exemplarymethanotrophic bacteria include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, orMethanomonas.

Methanotrophic bacteria are classified into three groups based on theircarbon assimilation pathways and internal membrane structure: type I(gamma proteobacteria), type II (alpha proteobacteria, and type X (gammaproteobacteria). Type I methanotrophs use the ribulose monophosphate(RuMP) pathway for carbon assimilation whereas type II methanotrophs usethe serine pathway. Type X methanotrophs use the RuMP pathway but alsoexpress low levels of enzymes of the serine pathway. Methanotrophicbacteria employed in the practice of the present invention includeobligate methanotrophs, which can only utilize C₁ substrates for carbonand energy sources, and facultative methanotrophs, which naturally havethe ability to utilize some multi-carbon substrates as a carbon andenergy source.

Exemplary facultative methanotrophs employed in the practice of thepresent invention include some species of Methylocella, Methylocystis,and Methylocapsa (e.g., Methylocella silvestris, Methylocella palustris,Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystisbryophila, and Methylocapsa aurea KYG), Methylobacterium organophilum(ATCC 27,886), Methylibium petroleiphilum, or high growth variantsthereof. Exemplary obligate methanotrophic bacteria useful in thepractice of the present invention include Methylococcus capsulatus Bath(NCIMB 11132), Methylomonas sp. 16a (ATCC PTA 2402), Methylosinustrichosporium OB3b (NRRL B-11,196), Methylosinus sporium (NRRLB-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonas methanica(NRRL B-11,199), Methylomonas albus (NRRL B-11,200), Methylobactercapsulatus Y (NRRL B-11,201), Methylomonas flagellata sp. AJ-3670 (FERMP-2400), Methylacidiphilum infernorum, Methylacidiphilum fumariolicum,Methylomicrobium alcaliphilum, Methyloacida kamchatkensis, or highgrowth variants thereof.

Suitable C₁ metabolizing microorganisms useful in the practice of thepresent invention include syngas metabolizing bacteria such as, forexample, Clostridium, Moorella, Pyrococcus, Eubacterium,Desulfobacterium, Carboxydothermus, Acetogenium, Acetobacterium,Acetoanaerobium, Butyribaceterium, Peptostreptococcus, and the like.Exemplary syngas metabolizing bacteria include Clostridiumautoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei,Clostridium carboxydivorans, Butyribacterium methylotrophicum,Clostridium woodii, Clostridium neopropanologen, and the like.

Other suitable C₁ metabolizing microorganisms useful in the practice ofthe present invention include eukaryotes such as, for example, yeast,including Candida, Yarrowia, Hansenula, Pichia, Torulopsis, Rhodotorula,and the like.

Each of the microorganisms of this disclosure may be grown as anisolated culture, with a heterologous organism that may aid with growth,or one or more of these bacteria may be combined to generate a mixedculture. The term “heterologous” when referring to an organism refers toa species that is different from the host cell. In still furtherembodiments, C₁ metabolizing non-photosynthetic microorganisms of thisdisclosure are obligate C₁ metabolizing non-photosyntheticmicroorganisms, such as an obligate methanotroph or methylotroph.

Any one of the aforementioned C₁ metabolizing microorganisms can be usedas a parent or reference host cell to make a recombinant C₁ metabolizingmicroorganisms of this disclosure. As used herein, “recombinant” refersto a non-naturally-occurring organism, microorganism, cell, nucleic acidmolecule, or vector that has at least one genetic alteration or has beenmodified by the introduction of a exogenous nucleic acid molecule, orrefers to a cell that has been altered such that the expression of anendogenous nucleic acid molecule or gene can be controlled. Recombinantalso refers to a cell that is derived from a cell or is progeny of acell having one or more such modifications. Genetic alterations include,for example, modifications introducing expressible nucleic acidmolecules encoding proteins or enzymes, or other nucleic acid moleculeadditions, deletions, substitutions or other functional alteration of acell's genetic material. For example, recombinant cells may expressgenes or other nucleic acid molecules that are not found in identicalform within the native cell (i.e., unmodified or wild type cell), or mayprovide an altered expression pattern of endogenous genes, such genesthat may otherwise be over-expressed, under-expressed, minimallyexpressed, or not expressed at all.

Any of the recombinant C₁ metabolizing microorganisms or methanotrophicbacteria described herein may be transformed to comprise at least oneexogenous nucleic acid to provide the host with a new or enhancedactivity (e.g., enzymatic activity) or may be genetically modified toremove or substantially reduce an endogenous gene function using any ofa variety of methods known in the art.

Transformation refers to the introduction of a nucleic acid molecule(e.g., exogenous nucleic acid molecule) into a host cell. Thetransformed host cell may carry the exogenous nucleic acid moleculeextra-chromosomally or integrated in the chromosome. Integration into ahost cell genome and self-replicating vectors generally result ingenetically stable inheritance of the transformed nucleic acid molecule.Host cells containing the transformed nucleic acid molecules arereferred to as “non-naturally occurring” or “genetically engineered” or“recombinant” or “transformed” or “transgenic” cells (e.g., bacteria).

Expression systems and expression vectors useful for the expression ofexogenous nucleic acids in C₁ metabolizing microorganisms (e.g.,methanotrophic bacteria) are known.

Electroporation of C₁ metabolizing bacteria is described herein and hasbeen previously described in, for example, Toyama et al., FEMSMicrobiol. Lett. 166:1, 1998; Kim and Wood, Appl. Microbiol. Biotechnol.48:105, 1997; Yoshida et al., Biotechnol. Lett. 23:787, 2001, and U.S.Patent Appl. Pub. No. 2008/0026005.

Bacterial conjugation, which refers to a particular type oftransformation involving direct contact of donor and recipient cells, ismore frequently used for the transfer of nucleic acid molecules into C₁metabolizing bacteria. Bacterial conjugation involves mixing “donor” and“recipient” cells together in close contact with each other. Conjugationoccurs by formation of cytoplasmic connections between donor andrecipient bacteria, with unidirectional transfer of newly synthesizeddonor nucleic acid molecules into the recipient cells. A recipient in aconjugation reaction is any cell that can accept nucleic acids throughhorizontal transfer from a donor bacterium. A donor in a conjugationreaction is a bacterium that contains a conjugative plasmid, conjugativetransposon, or mobilized plasmid. The physical transfer of the donorplasmid can occur through a self-transmissible plasmid or with theassistance of a “helper” plasmid. Conjugations involving C₁ metabolizingbacteria is described herein and have been previously described inStolyar et al., Mikrobiologiya 64:686, 1995; Motoyama et al., Appl.Micro. Biotech. 42:67, 1994; Lloyd et al., Arch. Microbiol. 171:364,1999; PCT Publication No. WO 02/18617; and Ali et al., Microbiol.152:2931, 2006.

Expression control sequences suitable for use in the practice of thepresent invention include, for example, promoters, terminators,enhancers, repressors, inducers, and the like. Promoters suitable foruse in the practice of the present invention may be constitutive, leaky,or inducible, and native or non-native to the host cell employed.Exemplary promoters include a pyruvate decarboxylase (PDC) a promoter, adeoxy-xylulose phosphate synthase promoter, a methanol dehydrogenasepromoter (MDH) (such as, for example, the promoter in the upstreamintergenic region of the mxaF gene from Methylococcus capsulatus Bath(Acc. No. MCA0779) or the MDH promoter from M. extorquens (See Springeret al., FEMS Microbiol. Lett. 160:119 (1998)), a hexulose 6-phosphatesynthase promoter, a ribosomal protein S16 promoter, a serinehydroxymethyl transferase promoter, a serine-glyoxylate aminotransferasepromoter, a phosphoenolpyruvate carboxylase promoter, a T5 promoter, Trcpromoter, a promoter for PHA synthesis (Foellner et al., Appl.Microbiol. Biotechnol. 40:284, 1993), a pyruvate decarboxylase promoter(Tokuhiro et al., Appl. Biochem. Biotechnol. 131:795, 2006), the lacoperon Plac promoter (Toyama et al., Microbiol. 143:595, 1997), a hybridpromoter such as Ptrc (Brosius et al., Gene 27:161, 1984), promotersidentified from native plasmid in methylotrophs (EP 296484),methanotrophs, and the like.

Additionally, suitable homologous or heterologous promoters for highexpression of exogenous nucleic acid molecules may be utilized. Forexample, U.S. Pat. No. 7,098,005 describes the use of promoters for highexpression in the presence of methane or methanol of a heterologouscoding nucleic acid in C₁ metabolizing bacteria.

In certain embodiments, regulated expression of exogenous nucleic acidsencoding a carbohydrate biosynthesis enzyme may be desirable to optimizegrowth rate of the non-naturally occurring C₁ metabolizing microorganismand may improve bacterial growth in a variety of carbon sourceconditions. This may be achieved through the use of an induciblepromoter system.

In certain embodiments, a nucleic acid encoding CB enzyme is operativelylinked to an inducible promoter. Inducible promoter systems employed inthe practice of the present invention include those known in the art andinclude tetracycline inducible promoter system; IPTG/lac operoninducible promoter system, heat shock inducible promoter system;metal-responsive promoter systems; nitrate inducible promoter system;light inducible promoter system; ecdysone inducible promoter system, theinducible/regulatable system described for use in methylotrophic andmethanotrophic bacteria (see, e.g., U.S. Patent Appl. No. US2010/0221813, which is incorporated herein by reference), and the like.For example, in one embodiment, the non-naturally occurring C₁metabolizing microorganism (e.g., methanotroph, methylotroph) comprises:(1) an exogenous nucleic acid encoding CB enzyme, operatively linked toa promoter flanked by lacO operator sequences, and (2) an exogenousnucleic acid encoding a lad repressor protein operatively linked to aconstitutive promoter (e.g., hexulose-6-phosphate synthase promoter).Induction is initiated when Lad repressor protein binds to lacO operatorsequences flanking the LDH or other promoter, preventing transcription.IPTG binds lad repressor and releases it from lacO sequences, allowingtranscription. By using an inducible promoter system, lactate synthesismay be controlled by the addition of an inducer.

The expression systems and expression vectors employed in the practiceof the present invention optionally contain genetic elements, such as,for example, one or more ribosome binding sites for translationinitiation and a transcription termination site, polyadenylationsignals, restriction enzyme sites, multiple cloning sites, other codingsegments, and the like. In certain embodiments, promoters and/or codonoptimization (described in more detail hereinabove) are used for highconstitutive expression of exogenous polynucleotides encoding one ormore carbohydrate biosynthesis enzymes in host methanotrophic bacteria.Regulated expression of an exogenous nucleic acid in a hostmethanotrophic bacterium may also be utilized. For example, aninducible/regulatable system of recombinant protein expression inmethylotrophic and methanotrophic bacteria as described in, for example,U.S. Patent Appl. No. US 2010/0221813 may be used.

Methods of Producing a Desired Carbohydrate

The present disclosure provides a method of producing a carbohydrate byculturing a recombinant C₁ metabolizing microorganism of the presentdisclosure in the presence of methane (from any source), or a naturalgas-derived carbon feedstock under conditions sufficient to produce thecarbohydrate. In a specific embodiment, the present disclosure providesa method of producing a carbohydrate by culturing a recombinant C₁metabolizing microorganism in the presence of a natural gas-derivedcarbon feedstock under conditions sufficient to produce thecarbohydrate, wherein the C₁ metabolizing microorganism comprises anexogenous nucleic acid encoding a carbohydrate biosynthesis enzyme.Typically, the natural gas-derived carbon feedstock is natural gas,methane, or syngas. Conditions for culturing exemplary C₁ metabolizingmicroorganisms are illustrated in Example 1.

In a further embodiment, the present disclosure provides a method ofproducing a carbohydrate, said method comprising culturing a recombinantC₁ metabolizing microorganism in the presence of methane underconditions sufficient to produce the carbohydrate, wherein the C₁metabolizing microorganism comprises an exogenous nucleic acid encodinga carbohydrate biosynthesis enzyme. In this embodiment, methane from anysource is suitable for use in the practice of the present invention,including natural gas, bio-methane, and the like. As used herein, theterm “bio-methane” refers to methane generated by fermentation oforganic matter such as, for example, manure, waste water sludge,municipal solid waste, and the like, under anaerobic conditions.

A variety of culture methodologies may be used for the microorganismsdescribed herein. For example, C₁ metabolizing microorganisms (such asmethanotroph or methylotroph bacteria) may be grown by batch culture orcontinuous culture methodologies. In certain embodiments, the culturesare grown in a controlled culture unit, such as a fermentor, bioreactor,hollow fiber cell, or the like. Generally cells in log phase are oftenresponsible for the bulk production of a product or intermediate ofinterest in some systems, whereas stationary or post-exponential phaseproduction can be obtained in other systems.

A classical batch culturing method is a closed system in which the mediacomposition is set when the culture is started and is not altered duringthe culture process. That is, media is inoculated at the beginning ofthe culturing process with one or more microorganisms of choice and thenare allowed to grow without adding anything to the system. As usedherein, a “batch” culture is in reference to not changing the amount ofa particular carbon source initially added, whereas control of factorssuch as pH and oxygen concentration can be monitored and altered duringthe culture. In batch systems, metabolite and biomass compositions ofthe system change constantly up to the time the culture is terminated.Within batch cultures, cells (e.g., bacteria such as methylotrophs) willgenerally move from a static lag phase to a high growth logarithmicphase to a stationary phase where growth rate is reduced or stopped (andwill eventually lead to cell death if conditions do not change).

A fed-batch system is a variation on the standard batch system in whicha carbon substrate of interest is added in increments as the cultureprogresses. Fed-batch systems are useful when cell metabolism is likelyto be inhibited by catabolite repression and when it is desirable tohave limited amounts of substrate in the media. Since it is difficult tomeasure actual substrate concentration in fed-batch systems, an estimateis made based on changes of measureable factors such as pH, dissolvedoxygen, and the partial pressure of waste gases. Batch and fed-batchculturing methods are common and known in the art (see, e.g., Thomas D.Brock, Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) Ed.(1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, Appl.Biochem. Biotechnol. 36:227, 1992).

Continuous cultures are “open” systems in the sense that defined culturemedia is continuously added to a bioreactor while an equal amount ofused (“conditioned”) media is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constant high,liquid phase density where cells are primarily in logarithmic growthphase. Alternatively, continuous culture may be practiced withimmobilized cells (e.g., biofilm) where carbon and nutrients arecontinuously added and valuable products, by-products, and wasteproducts are continuously removed from the cell mass. Cellimmobilization may be achieved with a wide range of solid supportscomposed of natural materials, synthetic materials, or a combinationthereof.

Continuous or semi-continuous culture allows for the modulation of oneor more factors that affect cell growth or end product concentration.For example, one method may maintain a limited nutrient at a fixed rate(e.g., carbon source, nitrogen) and allow all other parameters to changeover time. In other embodiments, several factors affecting growth may becontinuously altered while cell concentration, as measured by mediaturbidity, is kept constant. The goal of a continuous culture system isto maintain steady state growth conditions while balancing cell loss dueto media being drawn off against the cell growth rate. Methods ofmodulating nutrients and growth factors for continuous culture processesand techniques for maximizing the rate of product formation are wellknown in the art (see Brock, 1992).

Liquid phase bioreactors (e.g., stirred tank, packed bed, one liquidphase, two liquid phase, hollow fiber membrane) are well known in theart and may be used for growth of non-naturally occurring microorganismsand biocatalysis.

By using gas phase bioreactors, substrates for bioproduction areabsorbed from a gas by non-naturally occurring microorganisms, celllysates or cell-free fractions thereof, rather than from a liquid. Useof gas phase bioreactors with microorganisms is known in the art (e.g.,U.S. Pat. Nos. 2,793,096; 4,999,302; 5,585,266; 5,079,168; and6,143,556; U.S. Statutory Invention Registration H1430; U.S. PatentApplication Publication No. 2003/0032170; Emerging Technologies inHazardous Waste Management III, 1993, eds. Tedder and Pohland, pp411-428). Exemplary gas phase bioreactors include single pass system,closed loop pumping system, and fluidized bed reactor. By utilizing gasphase bioreactors, methane or other gaseous substrates are readilyavailable for bioconversion by polypeptides with, for example,monooxygenase activity. In certain embodiments, methods for converting agas into a carbohydrate are performed in gas phase bioreactors. Infurther embodiments, methods for converting a gas into a carbohydrateare performed in fluidized bed reactors. In a fluidized bed reactor, afluid (i.e., gas or liquid) is passed upward through particle bedcarriers, usually sand, granular-activated carbon, or diatomaceousearth, on which microorganisms can attach and grow. The fluid velocityis such that particle bed carriers and attached microorganisms aresuspended (i.e., bed fluidization). The microorganisms attached to theparticle bed carriers freely circulate in the fluid, allowing foreffective mass transfer of substrates in the fluid to the microorganismsand increased microbial growth. Exemplary fluidized bed reactors includeplug-flow reactors and completely mixed reactors. Uses of fluidized bedreactors with microbial biofilms are known in the art (e.g., Pfluger etal., Bioresource Technol. 102:9919, 2011; Fennell et al., Biotechnol,Bioengin. 40:1218, 1992; Ruggeri et al., Water Sci. Technol. 29:347,1994; U.S. Pat. Nos. 4,032,407; 4,009,098; 4,009,105; and 3,846,289).

Recombinant C₁ metabolizing microorganisms described in the presentdisclosure may be grown as an isolated pure culture, with a heterologousnon-C₁ metabolizing microorganism(s) that may aid with growth, or withone or more different strains or species of C₁ metabolizingmicroorganisms may be combined to generate a mixed culture.

In certain embodiments, carbohydrates of the present disclosure areproduced during a specific phase of cell growth (e.g., lag phase, logphase, stationary phase, or death phase). It may be desirable for carbonfrom feedstock to be converted to the carbohydrate rather than to growthand maintenance of C₁ metabolizing microorganism. In some embodiments,non-naturally occurring C₁ metabolizing microorganism (e.g.,methanotrophs, methylotrophs) as provided herein are cultured to a lowto medium cell density (OD₆₀₀) and then production of carbohydrate isinitiated. In some embodiments, a carbohydrate is produced whilemethanotrophic bacteria are no longer dividing or dividing very slowly.In some embodiments, the carbohydrate is produced only during stationaryphase. In some embodiments, the carbohydrate is produced during logphase and stationary phase.

The fermenter composition comprising the carbohydrate produced by arecombinant C₁ metabolizing microorganism (e.g., methanotrophs,methylotrophs) provided herein may further comprise other organiccompounds associated with biological fermentation processes. Forexample, biological by-products of fermentation may include one or moreof alcohols, epoxides, aldehydes, ketones, esters, or a combinationthereof. In certain embodiments, the fermenter composition may containone or more of the following alcohols: methanol, ethanol, butanol, orpropanol. Other compounds, such as H₂O, CO, CO₂, CO N₂, H₂, O₂, andunutilized carbon feedstocks, such as methane, ethane, propane, andbutane, may also be present in the fermenter off-gas.

In certain embodiments, the recombinant C₁ metabolizing microorganisms(e.g., methanotrophs, methylotrophs) provided herein produce acarbohydrate of the present invention at about 0.001 g/L of culture toabout 500 g/L of culture. In some embodiments, the amount ofcarbohydrate produced is about 1 g/L of culture to about 100 g/L ofculture. In some embodiments, the amount of carbohydrate produced isabout 0.001 g/L, 0.01 g/L, 0.025 g/L, 0.05 g/L, 0.1 g/L, 0.15 g/L, 0.2g/L, 0.25 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9g/L, 1 g/L, 2.5 g/L, 5 g/L, 7.5 g/L, 10 g/L, 12.5 g/L, 15 g/L, 20 g/L,25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L,90 g/L, 100 g/L, 125 g/L, 150 g/L, 175 g/L, 200 g/L, 225 g/L, 250 g/L,275 g/L, 300 g/L, 325 g/L, 350 g/L, 375 g/L, 400 g/L, 425 g/L, 450 g/L,475 g/L, or 500 g/L.

Products

The present disclosure provides other useful products in addition to therecombinant C₁ metabolizing cells described herein. In one embodiment,the present disclosure provides a biomass comprising a recombinant C₁metabolizing microorganism as described herein. In a specificembodiment, the present disclosure provides a biomass comprising arecombinant C₁ metabolizing microorganism, wherein the recombinant C₁metabolizing microorganism comprises an exogenous nucleic acid encodinga carbohydrate biosynthesis enzyme and wherein the recombinant C₁metabolizing microorganism is capable of converting a naturalgas-derived feedstock into a desired carbohydrate. In a specificembodiment, the exogenous nucleic acid encodes a β-glucan biosynthesisenzyme, for example, a β-(1,3)-glucan synthase. In some embodiments, thebiomass comprises a recombinant C₁ metabolizing microorganism and adesired carbohydrate, wherein the desired carbohydrate is a β-glucan andthe recombinant C₁ metabolizing microorganism comprises an exogenousnucleic acid encoding a β-glucan biosynthesis enzyme, and wherein the C₁metabolizing microorganism is capable of converting a naturalgas-derived feedstock into a β-glucan. Exemplary β-glucans include aβ-(1,3)-glucan, a β-(1,3)(1,6)-glucan, a β-(1,3)(1.4)-glucan, and aβ-(1,4)-glucan. In certain embodiments, the desired carbohydrate isselected from a β-(1,3)-glucan, a β-(1,3)(1,6)-glucan, or aβ-(1,3)(1.4)-glucan. In other embodiments, the desired carbohydrate is aβ-(1,3)-glucan.

In a further embodiment, the present disclosure provides a biomasscomprising a recombinant C₁ metabolizing microorganism, wherein therecombinant C₁ metabolizing microorganism comprises an exogenous nucleicacid encoding a carbohydrate biosynthesis enzyme and wherein therecombinant C₁ metabolizing microorganism is capable of convertingmethane into a desired carbohydrate. In a specific embodiment, theexogenous nucleic acid encodes a β-glucan biosynthesis enzyme, forexample, a β-(1,3)-glucan synthase, and the C₁ metabolizingmicroorganism is capable of converting methane into a β-glucan.Typically the β-glucan is selected from the group consisting of aβ-glucan, such as, for example, a β-(1,3)-glucan, a β-(1,3)(1,6)-glucan,a β-(1,3)(1,4)-glucan, and a β-(1,4)-glucan. In certain embodiments, thedesired carbohydrate is selected from the group consisting of aβ-(1,3)-glucan, a β-(1,3)(1,6)-glucan, a β-(1,3)(1,4)-glucan. In otherembodiments, the desired carbohydrate is a β-(1,3)-glucan.

As used herein, “biomass” refers to organic material having a biologicalorigin, which may include one or more of whole cells, lysed cells,extracellular material, or the like. For example, the material harvestedfrom a cultured microorganism (e.g., bacterial or yeast culture) isconsidered the biomass, which can include cells, cell membranes, cellcytoplasm, inclusion bodies, products secreted or excreted into theculture medium, or any combination thereof. In certain embodiments,biomass comprises the C₁ metabolizing microorganisms of this disclosuretogether with the media of the culture in which the C₁ metabolizingmicroorganisms of this disclosure were grown. In other embodiments,biomass comprises a C₁ metabolizing microorganisms (whole or lysed orboth) of this disclosure recovered from a culture grown on a C₁substrate (e.g., natural gas, methane, and the like). In still otherembodiments, biomass comprises the spent media supernatant from aculture of C₁ metabolizing microorganism cultured on a C₁ substrate.Such a culture may be considered a renewable resource. Biomass of thepresent invention is enriched with respect to levels of the desiredcarbohydrate.

Recombinant C₁ metabolizing microorganism of the present disclosure thatare provided with a natural gas-derived substrate for cell growth aredistinctive with respect to their carbon fingerprint as represented bytheir δ¹³C values (as are the products derived from such recombinant C₁metabolizing microorganisms). By way of background, stable isotopicmeasurements and mass balance approaches are widely used to evaluateglobal sources and sinks of methane (see Whiticar and Faber, Org.Geochem. 10:759, 1986; Whiticar, Org. Geochem. 16: 531, 1990). To useδ¹³C values of residual methane to determine the amount oxidized, it isnecessary to know the degree of isotopic fractionation caused bymicrobial oxidation of methane. For example, aerobic methanotrophs canmetabolize methane through a specific enzyme, methane monooxygenase(MMO). Methanotrophs convert methane to methanol and subsequentlyformaldehyde. Formaldehyde can be further oxidized to CO₂ to provideenergy to the cell in the form of reducing equivalents (NADH), orincorporated into biomass through either the RuMP or Serine cycles(Hanson and Hanson, Microbiol. Rev. 60:439, 1996), which are directlyanalogous to carbon assimilation pathways in photosynthetic organisms.More specifically, a Type I methanotroph uses the RuMP pathway forbiomass synthesis and generates biomass entirely from CH₄, whereas aType II methanotroph uses the serine pathway that assimilates 50-70% ofthe cell carbon from CH₄ and 30-50% from CO₂ (Hanson and Hanson, 1996).Methods for measuring carbon isotope compositions are provided in, forexample, Templeton et al. (Geochim. Cosmochim. Acta 70:1739, 2006),which methods are hereby incorporated by reference in their entirety.Examples 2 describes the characterization of stable carbon isotopedistribution in the cells of different C₁ metabolizing microorganisms.The highly negative δ¹³C values for the cells was similarly reflected inthe δ¹³C of compounds extracted from these cells, i.e., lipid fractions.The δ¹³C of the invention products described herein (i.e., a recombinantC₁ metabolizing microorganism of the present disclosure as describedherein), related biomass and carbohydrate compositions derivedtherefrom) can vary depending on the source and purity of the C₁substrate used as demonstrated in Example 2.

In certain embodiments, a recombinant C₁ metabolizing microorganism ofthe present disclosure, and related biomass and carbohydratecompositions derived therefrom, exhibit a δ¹³C of less than −30‰, lessthan −31‰, less than −32‰, less than −33‰, less than −34‰, less than−35‰, less than −36‰, less than −37‰, less than −38‰, less than −39‰,less than −40‰, less than −41‰, less than −42‰, less than −43‰, lessthan −44‰, less than −45‰, less than −46‰, less than −47‰, less than−48‰, less than −49‰, less than −50‰, less than −51‰, less than −52‰,less than −53‰, less than −54‰, less than −55‰, less than −56‰, lessthan −57‰, less than −58‰, less than −59‰, less than −60‰, less than−61‰, less than −62‰, less than −63‰, less than −64‰, less than −65‰,less than −66‰, less than −67‰, less than −68‰, less than −69‰, or lessthan −70‰.

In certain embodiments, a recombinant C₁ metabolizing microorganism ofthe present disclosure, and related biomass and carbohydratecompositions derived therefrom, exhibit a δ¹³C of about −35‰ to about−50‰, −45‰ to about −35‰, or about −50‰ to about −40‰, or about −45‰ toabout −65‰, or about −60‰ to about −70‰, or about −30‰ to about −70‰.

In further embodiments, a C₁ metabolizing non-photosyntheticmicroorganism biomass has a δ¹³C of less than about −30‰, or ranges fromabout −40‰ to about −60‰. In certain embodiments, the biomass comprisesa recombinant C₁ metabolizing non-photosynthetic microorganism togetherwith the spent media, or the biomass comprises a spent media supernatantcomposition from a culture of a recombinant C₁ metabolizingnon-photosynthetic microorganism, wherein the δ¹³C of the biomass isless than about −30‰. In certain other embodiments, the carbohydratecomposition is extracted or concentrated from a biomass, which cancomprise recombinant C₁ metabolizing non-photosynthetic microorganismstogether with the spent media from a culture, or a spent mediasupernatant composition from a culture of a recombinant C₁ metabolizingnon-photosynthetic microorganism.

In certain embodiments, a carbohydrate composition derived from a C₁metabolizing microorganism (which may optionally be an extract orisolate from the C₁ metabolizing microorganism biomass) compriseshydrogen, oxygen, and carbon atoms of at least about 50% to about 80% ofthe weight of the composition, and wherein the δ¹³C of the compositionis less than about −35‰ or less than about −36‰ or less than about −37‰or less than about −38‰ or less than about −39‰ or less than about −40‰.In certain embodiments, a carbohydrate composition derived therefromcomprises molecules having hydrogen, oxygen, and carbon atoms, whereinthe hydrogen, oxygen, and carbon atoms are at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%,or at least 90%, or at least 95% of the weight of the composition andwherein the δ¹³C of the composition ranges from about −30‰ to about−70‰, or wherein the δ¹³C in the biomass decreases as cell densityincreases by about −5‰ to about −20‰, or wherein the δ¹³C of the biomassis higher than that of CO₂ produced at the same time by an average of 5‰to 15‰ when cultured in the presence or absence of copper.

Typically, a carbohydrate composition comprises a polysaccharide, and insome instances, it comprises a monosaccharide. In other embodiments thecarbohydrate composition comprises a disaccharide. In some embodiments,the carbohydrate comprises a β-glucan. Typically, the β-glucan is aβ-(1,3)-glucan. In other embodiments, the β-glucan is aβ-(1,3)(1,6)-glucan, or a β-(1,3)(1,4)-glucan, or a β-(1,6)-glucan.Carbohydrate compositions derived from recombinant C₁ metabolizingmicroorganisms cultivated in the presence of a natural gas-derivedsubstrate exhibit the δ¹³C values described hereinabove.

Characterization of δ¹³C of some C₁ metabolizing microorganismscultivated in the presence of a natural gas-derived feedstock isillustrated in the examples, hereinbelow.

The present disclosure further provides an animal feed comprising therecombinant C₁ metabolizing microorganism, related biomass, and/orcarbohydrate composition of the present disclosure. As contemplated inthe practice of the present invention, the animal feed may be alivestock feed (such as, for example, pig feed, cattle feed, sheep feed,and the like), a poultry feed (such as, for example, chicken feed,turkey feed, and the like), or a fish feed (such as, for example, salmonfeed, shell fish feed, and the like). The animal feed may furthercomprise an additive, such as, for example, a plant-derived material(including, for example, those derived from grains such as, for example,corn, barley, oats, rice, rye, wheat, sorghum, Brewer's spent grain, andthe like; and those derived from legumes, such as, for example, alfalfa,clover, peas, beans, lentils, soybeans, and the like), an animal-derivedmaterial (such as, for example, fish meal), and/or amicroorganism-derived material (including, for example, biomass from aheterologous microorganism that may be, for example, a bacteria, ayeast, or an algae). In some embodiments, the plant-derived materialadditive is soy meal or pea protein,

In a further embodiment, the present disclosure provides a culture orfermentation medium comprising the recombinant C₁ metabolizingmicroorganism, related biomass, and/or carbohydrate composition of thepresent disclosure. Typically, the culture or fermentation mediumfurther comprises an amino acid and/or water. In an additionalembodiment, the present disclosure provides a cell culture compositioncomprising a culture or fermentation medium as described herein, and asecond microorganism. Typically, a second microorganism is a bacteria, ayeast, or an algae.

Embodiments of the present invention include the following:

1. A biomass derived from a culture of a recombinant C₁ metabolizingmicroorganism, wherein the recombinant microorganism comprises anexogenous nucleic acid encoding a carbohydrate biosynthesis enzyme,wherein the recombinant C₁ metabolizing is capable of converting anatural gas-derived carbon feedstock into a desired carbohydrate.

2. A biomass derived from a culture of a recombinant C₁ metabolizingmicroorganism, wherein the recombinant C₁ metabolizing microorganismcomprises an exogenous nucleic acid encoding a carbohydrate biosynthesisenzyme, wherein the recombinant C₁ metabolizing microorganism is capableof converting methane into a desired carbohydrate.

3. The biomass of any of embodiments 1-2, wherein the recombinant C₁metabolizing microorganism is a non-photosynthetic C₁ metabolizingmicroorganism.

4. The biomass of any of embodiments 1-3, wherein the carbohydrate isselected from the group consisting of a polysaccharide, a disaccharide,and a monosaccharide.

5. The biomass of embodiment 4, wherein the carbohydrate is amonosaccharide.

6. The biomass of embodiment 4, wherein the carbohydrate is adisaccharide.

7. The biomass of embodiment 4, wherein the carbohydrate is apolysaccharide.

8. The biomass of embodiment 7, wherein the polysaccharide is aβ-glucan.

9. The biomass of embodiment 8, wherein the β-glucan is β-(1,3)-glucan.

10. The biomass of embodiment 8, wherein the β-glucan isβ-(1,3)(1,6)-glucan.

11. The biomass of embodiment 8, wherein the β-glucan isβ-(1,3)(1,4)-glucan.

12. The biomass of embodiment 8, wherein the β-glucan is β-(1,4)-glucan.

13. The biomass of embodiment 8, wherein the β-glucan is β-(1,6)-glucan.

14. The biomass of any of embodiments 1-13, wherein the sequence of theexogenous nucleic acid is codon optimized for optimal expression fromthe recombinant C₁ metabolizing microorganism.

15. The biomass of any of embodiments 1-14, wherein the exogenousnucleic acid encodes a gluconeogenesis enzyme.

16. The biomass of embodiment 15, wherein the gluconeogenesis enzyme isselected from the group consisting of a pyruvate carboxylase, aphosphoenolpyruvate carboxykinase, an enolase, a phosphoglyceratemutase, a phosphoglycerate kinase, a glyceraldehyde-3-phosphatedehydrogenase, a Type A aldolase, a fructose 1,6-bisphosphatase, aphosphofructokinase, a phosphoglucose isomerase, a hexokinase, and aglucose-6-phosphate.

17. The biomass of any of embodiments 1-14, wherein the exogenousnucleic acid encodes a glycogenesis enzyme.

18. The biomass of embodiment 17, wherein the glycogenesis enzyme isselected from the group consisting of a glucose-1-phosphateadenyltransferase, a glycogen synthase, and a 1,4-alpha-glucan-branchingprotein.

19. The biomass of any of embodiments 8-14, wherein the exogenousnucleic acid is a β-glucan synthase.

20. The biomass of any of embodiments 1-19, wherein the exogenousnucleic acid encodes a carbohydrate biosynthesis enzyme that isendogenous to a bacteria.

21. The biomass of any of embodiments 1-19, wherein the exogenousnucleic acid encodes a carbohydrate biosynthesis enzyme that isendogenous to an organism selected from the group consisting of a yeast,a fungi, and a plant.

22. The biomass of any of embodiments 1-19, wherein the exogenousnucleic acid encodes a carbohydrate biosynthesis enzyme that isendogenous to a microorganism selected from the group consisting of E.coli and C. glutamicum.

23. The biomass of any of embodiments 1-14, wherein the exogenousnucleic acid encodes a carbohydrate biosynthesis enzyme selected fromthe group consisting of any of SEQ ID NOs:2, 4, 6, 8 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

24. The biomass of any of embodiments 1-23, wherein the exogenousnucleic acid encoding carbohydrate biosynthesis pathway enzyme isoperatively linked to an expression control sequence.

25. The biomass of embodiment 24, wherein the expression controlsequence is an exogenous expression control sequence.

26. The biomass of any of embodiments 1-25, wherein the C₁ metabolizingmicroorganism further comprises a deletion of an endogenous enzymeactivity. 27. The biomass according to any of embodiments 1-26, whereinthe C₁ metabolizing microorganism is a methanotroph.

28. The biomass according to embodiment 27, wherein the methanotroph isMethylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis,Methylomicrobium, Methanomonas, Methylocella, or Methylocapsa.

29. The biomass of embodiment 27, wherein the methanotroph is selectedfrom the group consisting of Methylococcus capsulatus Bath strain,Methylomonas methanica 16a (ATCC PTA 2402), Methylosinus trichosporiumOB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197),Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRLB-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus(NRRL B-11,201), Methylobacterium organophilum (ATCC 27,886),Methylomonas sp AJ-3670 (FERM P-2400), Methylocella silvestris,Methylocella palustris (ATCC 700799), Methylocella tundrae,Methylocystis daltona strain SB2, Methylocystis bryophila, Methylocapsaaurea KYG, Methylacidiphilum infernorum, Methylibium petroleiphilum, andMethylomicrobium alcahphilum.

30. The biomass according to any one of embodiments 1 and 3-29, whereinthe natural gas-derived carbon feedstock is selected from the groupconsisting of natural gas, syngas, methane, methanol, formaldehyde,formic acid, carbon monoxide, carbon dioxide, cyanide, a methylamine, amethylthiol, a methylhalogen, and any combination or two or morethereof.

31. The biomass of embodiment 30, wherein the natural gas-derived carbonfeedstock is natural gas.

32. The biomass of any of embodiments 1, and 3-30, wherein the naturalgas-derived carbon feedstock is methane.

33. The biomass of embodiment 30, wherein the natural gas-derived carbonfeedstock is syngas.

34. The biomass of embodiment 30, wherein the C₁ metabolizingmicroorganism is a syngas metabolizing bacteria.

35. The biomass according to embodiment 34, wherein the syngasmetabolizing bacteria is selected from the group consisting ofClostridiumautoethanogenum, Clostridium ljungdahli, Clostridiumragsdalei, Clostridium carboxydivorans, Butyridbacteriummethylotrophicum, Clostridium woodii, and Clostridium neopropanologen.

36. The biomass according to any one of embodiments 1 and 3-35, whereinthe δ¹³C of the biomass is less than −40‰.

37. The biomass of embodiment 2, wherein the methane is bio-methane.

38. A composition comprising a carbohydrate composition, wherein thecarbohydrate composition exhibits a δ¹³C of less than −40‰.

39. The composition of embodiment 38, wherein the carbohydrate comprisesa β-glucan.

40. The composition of embodiment 39, wherein the β-glucan isβ-(1,3)-glucan.

41. An animal feed comprising the biomass of any of embodiments 1-37 orthe composition of any of embodiments 38-40.

42. The animal feed of embodiment 41, further comprising a plant-derivedmaterial.

43. The animal feed of embodiment 41, wherein the plant-derived materialis selected from the group consisting of soybean meal and pea protein.

44. A culture or fermentation medium comprising the biomass of any ofembodiments 1-37 or the composition of any of embodiments 38-40.

45. A recombinant C₁ metabolizing microorganism, wherein the recombinantmicroorganism comprises an exogenous nucleic acid encoding acarbohydrate biosynthesis enzyme, wherein the recombinant C₁metabolizing microorganism is capable of converting a naturalgas-derived carbon feedstock into a desired carbohydrate.

46. A recombinant C₁ metabolizing microorganism, wherein the recombinantC₁ metabolizing microorganism comprises an exogenous nucleic acidencoding a carbohydrate biosynthesis enzyme, wherein the recombinant C₁metabolizing microorganism is capable of converting methane into adesired carbohydrate.

47. The recombinant C₁ metabolizing microorganism of any of embodiments45-46, wherein the recombinant C₁ metabolizing microorganism is anon-photosynthetic C₁ metabolizing microorganism.

48. The recombinant C₁ metabolizing microorganism of any of embodiments45-47, wherein the carbohydrate is selected from the group consisting ofa polysaccharide, a disaccharide, and a monosaccharide.

49. The recombinant C₁ metabolizing microorganism of embodiment 48,wherein the carbohydrate is a monosaccharide.

50. The recombinant C₁ metabolizing microorganism of embodiment 48,wherein the carbohydrate is a disaccharide.

51. The recombinant C₁ metabolizing microorganism of embodiment 48,wherein the carbohydrate is a polysaccharide.

52. The recombinant C₁ metabolizing microorganism of embodiment 51,wherein the polysaccharide is a β-glucan.

53. The recombinant C₁ metabolizing microorganism of embodiment 52,wherein the β-glucan is β-(1,3)-glucan.

54. The recombinant C₁ metabolizing microorganism of embodiment 52,wherein the β-glucan is β-(1,3)(1,6)-glucan.

55. The recombinant C₁ metabolizing microorganism of embodiment 52,wherein the β-glucan is β-(1,3)(1,4)-glucan.

56. The recombinant C₁ metabolizing microorganism of embodiment 52,wherein the β-glucan is β-(1,4)-glucan.

57. The recombinant C₁ metabolizing microorganism of embodiment 52,wherein the β-glucan is β-(1,6)-glucan.

58. The recombinant C₁ metabolizing microorganism of any of embodiments45-57, wherein the sequence of the exogenous nucleic acid is codonoptimized for optimal expression from the recombinant C₁ metabolizingmicroorganism.

59. The recombinant C₁ metabolizing microorganism of any of embodiments45-58, wherein the exogenous nucleic acid encodes a gluconeogenesisenzyme.

60. The recombinant C₁ metabolizing microorganism of embodiment 59,wherein the gluconeogenesis enzyme is selected from the group consistingof a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, anenolase, a phosphoglycerate mutase, a phosphoglycerate kinase, aglyceraldehyde-3-phosphate dehydrogenase, a Type A aldolase, a fructose1,6-bisphosphatase, a phosphofructokinase, a phosphoglucose isomerase, ahexokinase, and a glucose-6-phosphate.

61. The recombinant C₁ metabolizing microorganism of any of embodiments45-58, wherein the exogenous nucleic acid encodes a glycogenesis enzyme.

62. The recombinant C₁ metabolizing microorganism of embodiment 61,wherein the glycogenesis enzyme is selected from the group consisting ofa glucose-1-phosphate adenyltransferase, a glycogen synthase, and a1,4-alpha-glucan-branching protein.

63. The recombinant C₁ metabolizing microorganism of any of embodiments52-57, wherein the exogenous nucleic acid is a β-glucan synthase.

64. The recombinant C₁ metabolizing microorganism of any of embodiments45-63, wherein the exogenous nucleic acid encodes a carbohydratebiosynthesis enzyme that is endogenous to a bacteria.

65. The recombinant C₁ metabolizing microorganism of any of embodiments45-63, wherein the exogenous nucleic acid encodes a carbohydratebiosynthesis enzyme that is endogenous to an organism selected from thegroup consisting of a yeast, a fungi, and a plant.

66. The recombinant C₁ metabolizing microorganism of any of embodiments45-63, wherein the exogenous nucleic acid encodes a carbohydratebiosynthesis enzyme that is endogenous to a microorganism selected fromthe group consisting of E. coli, and C. glutamicum.

67. The recombinant C₁ metabolizing microorganism of any of embodiments45-57, wherein the exogenous nucleic acid encodes a carbohydratebiosynthesis enzyme selected from the group consisting of any of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,and 38.

68. The recombinant C₁ metabolizing microorganism of any of embodiments45-67, wherein the exogenous nucleic acid encoding carbohydratebiosynthesis pathway enzyme is operatively linked to an expressioncontrol sequence.

69. The recombinant C₁ metabolizing microorganism of embodiment 68,wherein the expression control sequence is an exogenous expressioncontrol sequence.

70. The recombinant C₁ metabolizing microorganism of any of embodiments45-69, wherein the C₁ metabolizing microorganism further comprises adeletion of an endogenous enzyme activity.

71. The recombinant C₁ metabolizing microorganism according to any ofembodiments 45-70, wherein the C₁ metabolizing microorganism is amethanotroph.

72. The recombinant C₁ metabolizing microorganism according toembodiment 71, wherein the methanotroph is Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, Methylocella, or Methylocapsa.

73. The recombinant C₁ metabolizing microorganism of embodiment 71,wherein the methanotroph is selected from the group consisting ofMethylococcus capsulatus Bath strain, Methylomonas methanica 16a (ATCCPTA 2402), Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinussporium (NRRL B-11,197), Methylocystis parvus (NRRL B-11,198),Methylomonas methanica (NRRL B-11,199), Methylomonas albus (NRRLB-11,200), Methylobacter capsulatus (NRRL B-11,201), Methylobacteriumorganophilum (ATCC 27,886), Methylomonas sp AJ-3670 (FERM P-2400),Methylocella silvestris, Methylocella palustris (ATCC 700799),Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystisbryophila, Methylocapsa aurea KYG, Methylacidiphilum infernorum,Methylibium petroleiphilum, and Methylomicrobium alcahphilum.

74. The recombinant C₁ metabolizing microorganism according to any oneof embodiments 45 and 47-73 wherein the natural gas-derived carbonfeedstock is selected from the group consisting of natural gas, syngas,methane, methanol, formaldehyde, formic acid, carbon monoxide, carbondioxide, cyanide, a methylamine, a methylthiol, a methylhalogen, and anycombination or two or more thereof.

75. The recombinant C₁ metabolizing microorganism of embodiment 74,wherein the natural gas-derived carbon feedstock is natural gas.

76. The recombinant C₁ metabolizing microorganism of embodiment 74,wherein the natural gas-derived carbon feedstock is methane.

77. The recombinant C₁ metabolizing microorganism of embodiment 74,wherein the natural gas-derived carbon feedstock is syngas.

78. The recombinant C₁ metabolizing microorganism of embodiment 77,wherein the C₁ metabolizing microorganism is a syngas metabolizingbacteria.

79. The biomass according to embodiment 78, wherein the syngasmetabolizing bacteria is selected from the group consisting ofClostridiumautoethanogenum, Clostridium ljungdahli, Clostridiumragsdalei, Clostridium carboxydivorans, Butyridbacteriummethylotrophicum, Clostridium woodii, and Clostridium neopropanologen.

80. The recombinant C₁ metabolizing microorganism according to any oneof embodiments 45 and 47-79, wherein the δ¹³C of the biomass is lessthan −40‰.

81. The recombinant C₁ metabolizing microorganism of embodiment 46,wherein the methane is bio-methane.

82. A method of producing a carbohydrate, said method comprisingculturing the recombinant C₁ metabolizing microorganism of any ofembodiments 45 and 47-68 in the presence of a natural gas-derived carbonfeedstock under conditions sufficient to produce the carbohydrate.

83. A method of producing a carbohydrate, said method comprisingculturing the recombinant C₁ metabolizing microorganism of embodiment 46in the presence of a methane under conditions sufficient to produce thecarbohydrate.

84. The method of embodiment 83, wherein the carbohydrate is a β-glucan.

85. A carbohydrate produced by the method of embodiment 82, wherein thecarbohydrate exhibits a δ¹³C in the range of from about −40‰ to about−60‰.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following, non-limiting examples.

EXAMPLES Example 1 Culture and Bioreactor Conditions for C₁ MetabolizingMicroorganisms

Exemplary C₁ metabolizing microorganisms of the instant disclosure(methanotrophs, methylotrophs, clostridia) were cultured in tubes, invials, in bottles, on plates, or in a bioreactor (fermentation). Growthconditions, media, and carbon source for various microorganisms aredescribed in this example.

Methylosinus trichosporium Strain OB3b (NCIMB 11131); Methylomonas sp.Strain 16a (ATCC PTA-2402); or Methylomonas methanica

For serum bottles, the bacteria were cultured at 30° C. in Higginsminimal nitrate salts medium (NSM; Cornish et al., J. Gen. Microbiol.130:2565, 1984; Park et al., Biotechnol. Bioeng. 38:423, 1991) or MM-W1medium. The headspace composition was adjusted to a 1:1 volume ofmethane:air. The bottles were shaken at a rate of 200-250 rpm.Alternatively, the culture was maintained on NSM-media plates containing1.5% w/v agar grown in a gas-tight chamber containing a 1:1 (v/v)methane:air gas mixture, or in the presence of methanol vapor (via 0.5mL methanol in the lid of parafilm-sealed plates) or on NSM-media platessupplemented with 0.5% methanol. Plates were incubated inverted in ahumidified chamber at 30° C.

The composition of the NSM medium used was as follows: 1.0 g MgSO₄*7H₂O,0.20 g CaCl₂*6H₂O, 2.0 ml chelated iron solution (0.1 g ferric (III)ammonium citrate or 0.5 g ferric (III) chloride; 0.2 g EDTA, sodiumsalt; 0.3 ml HCl, concentrated; 100.0 ml distilled deionized H₂O), 1.0 gKNO₃, 0.5 ml trace element solution (500.0 mg EDTA, 200.0 mg FeSO₄.7H₂O, 10.0 mg ZnSO₄*7H₂O, 3.0 mg MnCl₂*4H₂O, 30.0 mg H₃BO₃, 20.0 mgCoCl₂*6H₂O, 1.0 mg CaCl₂*2H₂O, 2.0 mg NiCl₂*6H₂O, 3.0 mg Na₂MoO₄*2H₂O,1.0 L distilled water), 0.272 g KH₂PO₄, 0.717 g Na₂HPO₄*12H₂O,optionally 12.5 g purified agar (e.g., Oxoid L28 or Bacto™ agar; usedwhen making plates), 1.0 L distilled deionized water, pH adjusted to 6.8and autoclaved at 121° C. for 15 minutes.

For fermentation, a 2-liter bioreactor containing 1 L of sterilizeddefined media MM-W1 was inoculated with cells from serum bottle batchcultures (10-20% v/v) grown in MM-W1 supplied with a 1:1 (v/v) mixtureof methane and air. The composition of medium MM-W1 used was as follows:0.8 mM MgSO₄*7H₂O, 10 mM NaNO₃, 0.14 mM CaCl₂, 1.2 mMNaHCO₃, 2.35 mMKH₂PO₄, 3.4 mM K₂HPO₄, 20.7 μMNa₂MoO₄*2H₂O, 1 μM CuSO₄*5H₂O, 10 μMFe^(III)-Na-EDTA, and 1 mL per liter of trace metals solution(containing, per liter 500 mg FeSO₄*7H₂O, 400 mg ZnSO₄*7H₂O, 20 mgMnCl₂*7H₂O, 50 mg CoCl₂*6H₂O, 10 mg NiCl₂*6H₂O, 15 mg H₃BO₃, 250 mgEDTA). Phosphate, bicarbonate, and Fe^(III)-Na-EDTA were added after themedia was autoclaved and cooled. Bicarbonate was added up to 0.1% (w/v)in certain fermentations. The reactor contents were stirred with anoverhead impeller at a constant 750 rpm. The culture was fed with aconstant methane sparging at about 60 mL/min to about 120 mL/min, whileconcentrated oxygen (at least 85%) was supplied at a variable rate ofabout 10-100 mL/min to maintain a dissolved oxygen level of about 40% toabout 80% (relative to air saturation of the media).

Temperature in the bioreactor was maintained at 30° C. and pH wasmaintained at 7.1±0.1 using automated addition of 0.5M NaOH and 0.5MHCl, along with other additions, to the culture about every 4 hours toabout 24 hours (corresponding to an OD₆₀₀ increase of approximately 5 ODunits). The other additions alternated between a metal addition (10 μMCuSO4, 5 μM FeSO4, 5 μM Fe^(III)-Na-EDTA final concentrations) and anutrient addition (5.75 mM KxHyPO4, 10 mM NaNO3). Under theseconditions, essentially linear growth was observed, with an effectivebiomass generation rate of about 2.7 to about 3.3 grams dry cell weightper liter per day to an OD₆₀₀ of greater than 20. Culture biomass washarvested by centrifugation, washed once in MM-W 1 media, and recoveredbiomass was either frozen at −80° C. or used immediately forfractionation of cellular components (e.g., lipid extraction).

A semi-continuous fermentation approach can also be applied to maintainbiomass productivity and reduce time associated with fermentationshut-down and start-up (i.e., turn-around time or lead time).

Harvesting of the bacterial biomass was performed at approximately 12-24hour intervals, as the culture density approached (but before entering)stationary phase. Approximately half of the bioreactor volume wasremoved by transferring to a separate container via centrifugal pump. Anequal volume of sterilized or recycled media was then returned to thebioreactor such that the optical density of the reactor wasapproximately half of its initial value. The bioreactor fermentation wascontinued according to the above protocol so that multiple cycles ofgrowth and biomass recovery could be carried out during a singlefermentation run.

Methylococcus capsulatus Bath (NCIMB 11132)

The bacteria were cultured at 42° C. in serum bottles containing Higginsminimal nitrate salts medium (NSM) or MM-W1 medium. The headspacecomposition was adjusted to a 1:1 volume of methane:air. The bottleswere shaken at a rate of 200-250 rpm. Alternatively, the culture wasmaintained on NSM-media plates solidified with 1.5% w/v agar grown in agas-tight chamber containing a 1:1 (v/v) methane:air gas mixture. Plateswere incubated inverted in the chamber at 42° C.

For fermentation, a 3-liter bioreactor containing 1.25 L sterilizedmedia MMF1.1 was inoculated with cells from serum bottle batch cultures(10-20% v/v) grown in the same media supplied with a 1:1 (v/v) mixtureof methane and air. The composition of medium MMF1.1 was as follows: 0.8mM MgSO₄*7H₂O, 40 mM NaNO₃, 0.14 mM CaCl₂, 6 mM NaHCO₃, 4.7 mM KH₂PO₄,6.8 mM K₂HPO₄, 20.7 μM Na2MoO₄*2H₂O, 6 μM CuSO₄*5H₂O, 10 μMFe^(III)-Na-EDTA, and 1 mL per liter of trace metals solution(containing, per liter 500 mg FeSO₄*7H₂O, 400 mg ZnSO₄*7H₂O, 20 mgMnCl₂*7H₂O, 50 mg CoCl₂*6H₂O, 10 mg NiCl₂*6H₂O, 15 mg H₃BO₃, 250 mgEDTA). Phosphate, bicarbonate, and Fe^(III)-Na-EDTA were added aftermedia was autoclaved and cooled. The reactor contents were stirred withan overhead impeller at a constant 750 rpm. The culture was fed with aconstant methane sparging at about 60 to about 200 mL/min, whileconcentrated oxygen (>85%) was supplied at a variable rate of 15-90mL/min and the dissolved oxygen level was maintained below 10% (relativeto air saturation of the media).

Temperature in the bioreactor was maintained at 44° C. and pH wasmaintained at 7.0±0.1 using automated addition of 0.5M NaOH and 0.5MHCl, along with additions of copper and iron (5 μM CuSO4, 5 μM FeSO₄, 10μM Fe^(III)-Na-EDTA final concentration) to the culture every 3-6 hours(corresponding to an OD₆₀₀ increase of approximately 3-5 OD units afterreaching OD 5). Under these conditions, essentially linear growth wasobserved, with effective biomass generation rate of more than 5 gramsdry cell weight per liter per day to an OD₆₀₀ of greater than 10.Culture biomass was harvested by centrifugation, the cells washed oncein MM-W1 media and cell pellets were either frozen at −80° C. or usedimmediately for fractionation of cellular components.

Nutrient depletion was recognized as an issue that could limit thegrowth yield during fermentation. To avoid limitation of nutrients,mainly nitrogen and phosphate, nutrient feeds composed of 2-foldconcentrated MMF1.1 were initiated after culture OD₆₀₀ exceeded 5. Thenutrient feed was initiated at dilution rates corresponding toapproximately half of the cultures' growth rate to avoid wash-out and tomaintain an increase in OD while expanding the culture volume. Thebioreactor fermentation was continued according to the above protocol sothat multiple cycles of growth and biomass recovery could be carried outduring a single fermentation run.

Methylobacterium extorquens or Methylosinus trichosporium Strain OB3b(NCIMB 11131)

The bacteria is cultured at 30° C. in tubes containing Higgins minimalnitrate salts medium (NSM) supplemented with 0.5% methanol. The tubesare shaken at a rate of 200-250 rpm. Alternatively, the cultures aremaintained on NSM-media plates containing 1.5% w/v agar grown in thepresence of methanol vapor (via 0.5 mL methanol in the lid ofparafilm-sealed plates) or supplemented with 0.5% methanol. Plates areincubated inverted in a humidified chamber under normal atmosphere at30° C.

For fermentation, a 2-liter bioreactor containing 1 L defined mediaMM-W1 is inoculated with cells from culture tube batch culture (10-20%v/v). The composition of medium MM-W1 was as described above. Thereactor contents are stirred with an overhead impeller at a constant 800rpm. The culture is fed with an initial bolus of methanol to a finalconcentration of 0.5% and variable methanol feed, while pure oxygen wassupplied at a variable rate of 30-100 mL/min to maintain a dissolvedoxygen level of 60-90% (relative to air saturation of the media).

Temperature in the bioreactor was maintained at 30° C. and pH wasmaintained at 7.1±0.1 using automated addition of 0.5M NaOH and 1M HCl,along with the metal and nutrient additions as described above. Underthese conditions, essentially linear growth is observed, with effectivebiomass generation rate 2.7 to 3.3 grams dry cell weight per liter perday to an OD₆₀₀ of greater than 20. Culture biomass was harvested bycentrifugation, the cells washed once in MM-W 1 media and cell pelletswere either frozen at −80° C. or used immediately for fractionation ofcellular components.

A semi-continuous fermentation approach can also be applied to maintainbiomass productivity and reduce time associated with fermentationshut-down and start-up (i.e., turn-around time or lead time).

Harvesting of the accumulated bacterial biomass was performed atapproximately 12-24 hour intervals, as the culture density approached(but before entering) stationary phase. Approximately half of thebioreactor volume was removed by transferring to a separate containervia centrifugal pump. An equal volume of fresh or recycled media wasthen returned to the bioreactor such that the optical density of thereactor was approximately half of its initial value. The bioreactorfermentation was continued according to the above protocol so thatmultiple cycles of growth and biomass recovery was carried out during asingle fermentation run.

Clostridium autoethanogenum and Clostridium ljungdahlii

The Clostridium bacteria are cultivated anaerobically in 100 mL modifiedPETC medium (ATCC medium 1754) at 37° C. in plastic-coated 500 ml-SchottDuran® GL45 bottles with butyl rubber stoppers and 200 kPa steel millwaste gas. Growth is monitored by measuring the optical density at 600nm (OD₆₀₀).

The modified PETC medium contains (per liter) 1 g NH₄Cl, 0.4 g KCl, 0.2g MgSO₄*7 H₂O, 0.8 g NaCl, 0.1 g KH₂PO₄, 20 mg CaCl₂*2 H₂O, 10 ml traceelements solution (see below), 10 ml Wolfe's vitamin solution (seebelow), 2 g NaHCO₃, and 1 mg resazurin. After the pH is adjusted to 5.6,the medium is boiled, dispensed anaerobically, and autoclaved at 121° C.for 15 min. Steel mill waste gas (composition: 44% CO, 32% N₂, 22% CO₂,2% H₂) or equivalent synthetic mixtures are used as a carbon source. Themedia has a final pH of 5.9 and is reduced with cysteine-HCl and Na₂S ata concentration of 0.008% (w/v).

The trace elements solution contains 2 g nitrilotriacetic acid (adjustedto pH 6 with KOH before addition of the remaining ingredients), 1 gMnSO₄, 0.8 g Fe(SO₄)₂(NH₄)₂*6 H₂O, 0.2 g CoCl₂*6 H₂O, 0.2 mg ZnSO₄*7H₂O, 20 mg CuCl₂*2 H₂O, 20 mg NiCl₂*6 H₂O, 20 mg Na₂MoO₄*2 H₂O, 20 mgNa₂SeO₄, and 20 mg Na₂WO₄ per liter.

Wolfe's vitamin solution (Wolin et al., J. Biol. Chem. 238:2882, 1963)contains (per liter) 2 mg biotin, 2 mg folic acid, 10 mg pyridoxinehydrochloride, 5 mg thiamine-HCl, 5 mg riboflavin, 5 mg nicotinic acid,5 mg calcium D-(+)-pantothenate, 0.1 mg vitamin B12, 5 mg p-aminobenzoicacid, and 5 mg thioctic acid.

a. Clostridium autoethanogenum Fermentation

Fermentation of Clostridium autoethanogenum is conducted using methodssimilar to those described in, for example, U.S. Patent Appl. No.2011/0300593. Briefly, a 2-liter bioreactor containing 1.3 L Solution A(3.083 g NH₄Ac; 0.61 g MgCl₂*6H₂O; 0.294 g CaCl₂*2H₂O; 0.15 g KCl; 0.12g NaCl (optional); up to 1 L with distilled water) is sparged with N₂gas. An 85% solution of H₃PO₄ (2.025 mL, 30 mM) is added and the pHadjusted to 5.3 using concentrated, aqueous NH₄OH. Then 13.5 mL SolutionB (20.0 mg Biotin; 20.0 mg Folic acid; 10.0 mg pyridoxine HCl; 50.0 mgthiamine*HCl; 50.0 mg Riboflavin; 50.0 mg nicotinic acid; 50.0 mgcalcium D-(*)-pantothenate; 50.0 mg vitamin B12; 50.0 mg p-aminobenzoicacid; 50.0 mg thioctic acid; up to 1 L with distilled water) is addedand the solution sparged with N₂ gas. Chromium (II) chloride is addeduntil the oxidation-reduction potential (ORP) of the solution decreasesto approximately −200 mV, wherein resazurin (1.35 mL of a 2 g/Lsolution) is added. Sodium polysulfide (5.4 mL of a 3M solution, seebelow) is added and the solution sparged with N₂ and then CO containinggas (1% H₂; 13% N₂; 71% CO; 15% CO₂). A metal sulfide solution (150 mL,see below) is added and the solution sparged a further 30 minutes,before inoculation with an actively growing C. autoethanogenum cultureat a level of approximately 5% (v/v).

The sodium polysulfide solution is prepared in a 500 ml flask that ischarged with Na₂S (93.7 g, 0.39 mol) and 200 ml H₂O. The solution isstirred until the salt dissolves and sulfur (25 g, 0.1 mol) is addedunder constant N₂ flow. After stirring at room temperature for 2 hours,the sodium polysulfide solution (about 4 M with respect to Na and about5 M with respect to sulfur), now a clear reddish brown liquid, istransferred into N₂ purged serum bottles, and wrapped in aluminum foil.

The chromium (II) solution is prepared in a 1 L three necked flask thatis fitted with a gas tight inlet and outlet to allow working under inertgas and subsequent transfer of the desired product into a suitablestorage flask. The flask is charged with CrCl₃*6 H₂O (40 g, 0.15 mol),zinc granules [20 mesh] (18.3 g, 0.28 mol), mercury (13.55 g, 1 mL,0.0676 mol) and 500 mL distilled water. Following flushing with N₂ forone hour, the mixture is warmed to about 80° C. to initiate thereaction. Following two hours of stirring under a constant N₂ flow, themixture is cooled to room temperature and continuously stirred foranother 48 hours by which time the reaction mixture turns into a deepblue solution. The solution is transferred into N₂ purged serum bottlesand stored at 4° C. for future use.

The metal sulfide solution is prepared by adding about 950 mL Solution Ainto a 1 L fermenter and sparging with N₂ gas. An 85% solution of H₃PO₄(1.5 mL, 30 mM) is added and the pH adjusted to 5.3 using concentratedaqueous NH₄OH. Solution B (10 mL) is added and the solution sparged withN₂. Chromium (II) chloride is added until the oxidation-reductionpotential (ORP) of the solution decreases to approximately −200 mV,wherein resazurin (1 mL of a 2 g/L solution) is added. Solution C (1/10;10 ml FeCl₃; 5 ml CoCl₂; 5 ml NiCl₂; 1 ml H₃BO₃; 1 ml Na₂MoO₄; 1 mlMnCl₂; 1 ml Na₂WO₄; 1 ml ZnCl₂; 1 ml Na₂SeO₃; into 1 L media) is added,then sodium polysulfide (2 mL of a 3M solution) is added, and then thesolution is sparged with N₂ gas.

Fermentation of a substrate comprising CO by C. autoethanogenum underbatch conditions in the presence of polysulfide results in asubstantially increased rate of accumulation and a final biomassaccumulation of approximately 4 g/L over a 2-3 day period. For example,following a short lag phase of approximately 1 day, the biomass canincrease from about 0.5 g/L up to at least 3.5 g/L over approximately 36hours of fermentation. Furthermore, acetate is not produced during thegrowth phase in the presence of polysulfide (as is typically found inbatch fermentations) and in certain circumstances some of the acetate isconsumed, such that there is a net decrease in the amount of acetate inthe fermenter. Culture biomass was harvested by centrifugation, thecells washed once in media and cell pellets were either frozen at −80°C. or used immediately for fractionation of cellular components.

A semi-continuous fermentation approach can also be applied to maintainbiomass productivity and reduce time associated with fermentationshut-down and start-up (i.e., turn-around time or lead time).

Harvesting of the accumulated bacterial biomass was performed atapproximately 12-24 hour intervals, as the culture density approached(but before entering) stationary phase. Approximately half of thebioreactor volume was removed by transferring to a separate containervia centrifugal pump. An equal volume of fresh or recycled media wasthen returned to the bioreactor such that the optical density of thereactor was approximately half of its initial value. The bioreactorfermentation was continued according to the above protocol so thatmultiple cycles of growth and biomass recovery was carried out during asingle fermentation run.

b. Clostridium ljungdahlii Fermentation

Fermentation of Clostridium ljungdahlii is performed using similarmethods to those described in, for example, U.S. Pat. Nos. 5,173,429 and5,593,886. Briefly, batch fermentations are conducted using abiologically pure culture of C. ljungdahlii. Preparation of the medium((1) 80.0 mL of a salt comprising KH₂PO₄ 3.00 g/L, K₂HPO₄ 3.00 g/L,(NH₄)₂SO₄ 6.00 g/L, NaCl 6.00 g/L, MgSO₄*2H₂O 1.25 g/L; (2) 1.0 g ofyeast extract; (3) 1.0 g of trypticase; (4) 3.0 ml of PFN (Pfenning)trace metal solution comprising FeCl₂*4H₂O 1500 mg, ZnSO₄*7H₂O 100 mg,MnCl₂*4H₂O 30 mg, H₃BO₃ 300 mg, CoCl₂*6H₂O 200 mg, CuCl₂*H₂O 10 mg,NiCl₂*6H₂O 20 mg, NaMoO₄*2H₂O 30 mg, Na_(z) SeO₃ 10 mg, and distilledwater up to 1 L; (5) 10.0 ml of B vitamins comprising Pyridoxal HCl 10mg, Riboflavin 50 mg, Thiamine HCl 50 mg, Nictotinic acid 50 mg,Ca-D-Pantotheinate 50 mg, Lipoic acid 60 mg, p-aminobenzoic acid 50 mg,Folic acid 20 mg, Biotin 20 mg, cyanocobalamin 50 mg, and distilledwater up to 1 L; (6) 0.5 g of cysteine HCl; (7) 0.06 g CaCl₂*2H₂O; (8)2.0 g NaHCO₃; (9) 1.0 mL resazurin (0.01%); and (10) 920.0 mL distilledwater) is carried out anaerobically in an atmosphere of 80% nitrogen and20% CO₂. The pH of the medium is controlled during fermentation andmaintained at 5.0 with HCl. If required, adjustments to the pH are madewith sterile 10% NaOH or 1.0% acetic acid solution. The medium istransferred to 157.5 mL serum bottles and sealed with butyl rubberstoppers and aluminum seals. The bottles are then autoclaved at 121° C.for 20 minutes.

Approximately 48 hours before commencing the experiment, a seed cultureis prepared from a stock culture of the C. ljungdahlii in a bottlesimilar to those as described above. The seed culture is grown in ashaker incubator at 37° C. and shaken at 100 rpm. Reducing solutions(2.0 ml Na₂S, 2.5% solution and 2.0 ml cysteine-HCl, 3.5% solution) areadded to the culture, which is placed in the shaker incubator forapproximately 15 minutes to allow for complete oxygen removal andtemperature acclimation. Unlike the procedure used for isolating abiologically pure culture of the organism, addition of methaneinhibitors is not required in batch fermentations.

Fermentation with C. ljungdahlii is performed in a New BrunswickScientific Bioflow IIc 2.5-liter fermenter containing nutrient media at37° C., and a constant fluid level of 1.5 liters is maintained while thefluid is agitated at variable rates of up to 1,000 revolutions perminute with gas introduced at a rate of approximately 500 cubiccentimeters per minute. Optimal gas retention times are in the range ofthree minutes. The gas feed is varied with its uptake by the bacteria,which is in turn a function of the cell density.

Harvesting of the accumulated bacterial biomass was performed atapproximately 12-24 hour intervals, as the culture density approached(but before entering) stationary phase. Approximately half of thebioreactor volume was removed by transferring to a separate containervia centrifugal pump. An equal volume of fresh or recycled media wasthen returned to the bioreactor such that the optical density of thereactor was approximately half of its initial value. The bioreactorfermentation was continued according to the above protocol so thatmultiple cycles of growth and biomass recovery was carried out during asingle fermentation run.

Example 2 Stable Carbon Isotope Distribution in Lipids from C₁Metabolizing Microorganisms

Dry samples of M. trichosporium biomass and lipid fractions wereanalyzed for carbon and nitrogen content (% dry weight), and carbon(¹³C) and nitrogen (′⁵N) stable isotope ratios via elementalanalyzer/continuous flow isotope ratio mass spectrometry using a CHNOSElemental Analyzer (vario ISOTOPE cube, Elementar, Hanau, Germany)coupled with an IsoPrime100 IRMS (Isoprime, Cheadle, UK). Samples ofmethanotrophic biomass cultured in fermenters or serum bottles werecentrifuged, resuspended in deionized water and volumes corresponding to0.2-2 mg carbon (about 0.5-5 mg dry cell weight) were transferred to 5×9mm tin capsules (Costech Analytical Technologies, Inc., Valencia,Calif.) and dried at 80° C. for 24 hours. Similarly, previouslyextracted lipid fractions were suspended in chloroform and volumescontaining 0.1-1.5 mg carbon were transferred to tin capsules andevaporated to dryness at 80° C. for 24 hours. Standards containing 0.1mg carbon provided reliable δ¹³C values.

The isotope ratio is expressed in “delta” notation (‰), wherein theisotopic composition of a material relative to that of a standard on aper million deviation basis is given by δ¹³C (orδ¹⁵N)=(R_(Sample)/R_(Standard-1))×1,000, wherein R is the molecularratio of heavy to light isotope forms. The standard for carbon is theVienna Pee Dee Belemnite (V-PDB) and for nitrogen is air. The NIST(National Institute of Standards and Technology) proposed SRM (StandardReference Material) No. 1547, peach leaves, was used as a calibrationstandard. All isotope analyses were conducted at the Center for StableIsotope Biogeochemistry at the University of California, Berkeley.Long-term external precision for C and N isotope analyses is 0.10‰ and0.15‰, respectively.

M. trichosporium strain OB3b was grown on methane in three differentfermentation batches, M capsulatus Bath was grown on methane in twodifferent fermentation batches, and Methylomonas sp. 16a was grown onmethane in a single fermentation batch. The biomass from each of thesecultures was analyzed for stable carbon isotope distribution (δ¹³Cvalues; see Table 3).

TABLE 3 Stable Carbon Isotope Distribution in Different MethanotrophsMethanotroph Batch No. EFT (h)† OD₆₀₀ DCW* δ¹³C Cells Mt OB3b 68A 481.80 1.00 −57.9 64 1.97 1.10 −57.8 71 2.10 1.17 −58.0 88 3.10 1.73 −58.197 4.30 2.40 −57.8 113 6.00 3.35 −57.0 127 8.40 4.69 −56.3 Mt OB3b 68B32 2.90 1.62 −58.3 41 4.60 2.57 −58.4 47 5.89 3.29 −58.0 56 7.90 4.41−57.5 Mt OB3b 68C 72 5.32 2.97 −57.9 79.5 5.90 3.29 −58.0 88 5.60 3.12−57.8 94 5.62 3.14 −57.7 Mc Bath 62B 10 2.47 0.88 −59.9 17.5 5.80 2.06−61.0 20 7.32 2.60 −61.1 23 9.34 3.32 −60.8 26 10.30 3.66 −60.1 Mc Bath62A 10 2.95 1.05 −55.9 13.5 3.59 1.27 −56.8 17.5 5.40 1.92 −55.2 23 6.082.16 −57.2 26 6.26 2.22 −57.6 Mms 16a 66B 16 2.13 0.89 −65.5 18 2.591.09 −65.1 20.3 3.62 1.52 −65.5 27 5.50 2.31 −66.2 40.5 9.80 4.12 −66.3*DCW, Dry Cell Weight is reported in g/L calculated from the measuredoptical densities (OD₆₀₀) using specific correlation factors relating ODof 1.0 to 0.558 g/L for Mt OB3b, OD of 1.0 to 0.355 g/L for Me Bath, andOD of 1.0 to 0.42 g/L for Mms 16a. For Mt OB3b, the initialconcentration of bicarbonate used per fermentation was 1.2 mM or 0.01%(Batch No. 68C) and 0.1% or 12 mM (Batch Nos. 68A and 68B). †EFT =effective fermentation time in hours

In addition, stable carbon isotope analysis was performed for biomassand corresponding lipid fractions (see Table 4) from strainsMethylosinus trichosporium OB3b (Mt OB3b), Methylococcus capsulatus Bath(Mc Bath), and Methylomonas sp. 16a (Mms 16a) grown on methane inbioreactors as described in Example 1.

TABLE 4 Stable Carbon Isotope Distribution in Cells and Lipids Batch No.Strain δ¹³C Cells δ¹³C Lipids 68C Mt OB3b −57.7 −48.6 62A Mc Bath −57.6−52.8 66A Mms 16a −64.4 −42.2

Biomass from strains Mt OB3b, Mc Bath and Mms 16a were harvested at 94 h(3.14 g DCW/L), 26 h (2.2 g DCW/L) and 39 h (1.14 g DCW/L),respectively. The δ¹³C values for lipids in Table 4 represent an averageof duplicate determinations.

Example 3 Effect of Methane Source and Purity on Stable Carbon IsotopeDistribution in Lipids

To examine methanotroph growth on methane containing natural gascomponents, a series of 0.5-liter serum bottles containing 100 mLdefined media MMS1.0 were inoculated with Methylosinus trichosporiumOB3b or Methylococcus capsulatus Bath from a serum bottle batch culture(5% v/v) grown in the same media supplied with a 1:1 (v/v) mixture ofmethane and air. The composition of medium MMS1.0 was as follows: 0.8 mMMgSO₄*7H₂O, 30 mM NaNO₃, 0.14 mM CaCl₂, 1.2 mM NaHCO₃, 2.35 mM KH₂PO₄,3.4 mM K₂HPO₄, 20.7 μM Na₂MoO₄*2H₂O, 6 μM CuSO₄*5H₂O, 10 μMFe^(III)-Na-EDTA, and 1 mL per liter of a trace metals solution(containing, per L: 500 mg FeSO4*7H₂O, 400 mg ZnSO₄*7H₂O, 20 mgMnCl₂*7H2O, 50 mg CoCl₂*6H₂O, 10 mg NiCl₂*6H₂O, 15 mg H₃BO₃, 250 mgEDTA). Phosphate, bicarbonate, and Fe^(III)-Na-EDTA were added aftermedia was autoclaved and cooled. The final pH of the media was 7.0±0.1.

The inoculated bottles were sealed with rubber sleeve stoppers andinjected with 60 mL methane gas added via syringe through sterile 0.45μm filter and sterile 27G needles. Duplicate cultures were each injectedwith 60 mL volumes of (A) methane of 99% purity (grade 2.0, Praxairthrough Alliance Gas, San Carlos, Calif.), (B) methane of 70% purityrepresenting a natural gas standard (Sigma-Aldrich; also containing 9%ethane, 6% propane, 3% methylpropane, 3% butane, and other minorhydrocarbon components), (C) methane of 85% purity delivered as a 1:1mixture of methane sources A and B; and (D) >93% methane (grade 1.3,Specialty Chemical Products, South Houston, Tex.; in-house analysisshowed composition >99% methane). The cultures were incubated at 30° C.(M trichosporium strain OB3b) or 42° C. (M capsulatus Bath) with rotaryshaking at 250 rpm and growth was measured at approximately 12 hourintervals by withdrawing 1 mL samples to determine OD₆₀₀. At thesetimes, the bottles were vented and headspace replaced with 60 mL of therespective methane source (A, B, C, or D) and 60 mL of concentratedoxygen (at least 85% purity). At about 24 hour intervals, 5 mL sampleswere removed, cells recovered by centrifugation (8,000 rpm, 10 minutes),and then stored at −80° C. before analysis.

Analysis of carbon and nitrogen content (% dry weight), and carbon (¹³C)and nitrogen (¹⁵N) stable isotope ratios, for methanotrophic biomassderived from M trichosporium strain OB3b and M capsulatus Bath werecarried out. Table 5 shows the results of stable carbon isotope analysisfor biomass samples from M capsulatus Bath grown on methane havingdifferent levels of purity and in various batches of bottle cultures.

TABLE 5 Stable Carbon Isotope Distribution of M. capsulatus Bath Grownon Different Methane Sources having Different Purity Methane* Batch No.Time (h)† OD₆₀₀ DCW (g/L) δ¹³C Cells A 62C 22 1.02 0.36 −40.3 56 2.010.71 −41.7 73 2.31 0.82 −42.5 62D 22 1.14 0.40 −39.3 56 2.07 0.73 −41.673 2.39 0.85 −42.0 B 62E 22 0.47 0.17 −44.7 56 0.49 0.17 −45.4 73 0.290.10 −45.4 62F 22 0.62 0.22 −42.3 56 0.63 0.22 −43.6 73 0.30 0.11 −43.7C 62G 22 0.70 0.25 −40.7 56 1.14 0.40 −44.8 73 1.36 0.48 −45.8 62H 220.62 0.22 −40.9 56 1.03 0.37 −44.7 73 1.23 0.44 −45.9 *Methane purity:A: 99% methane, grade 2.0 (min. 99%); B: 70% methane, natural gasstandard (contains 9% ethane, 6% propane, 3% methylpropane, 3% butane);C: 85% methane (1:1 mix of A and B methane) †Time = bottle culture timein hours

The average δ¹³C for M. capsulatus Bath grown on one source of methane(A, 99%) was −41.2±1.2, while the average δ¹³C for M. capsulatus Bathgrown on a different source of methane (B, 70%) was −44.2±1.2. Whenmethane sources A and B were mixed, an intermediate average δ¹³C of−43.8±2.4 was observed. These data show that the δ¹³C of cell materialgrown on methane sources A and B are significantly different from eachother due to the differences in the δ¹³C of the input methane. But,cells grown on a mixture of the two gasses preferentially utilize ¹²Cand, therefore, show a trend to more negative δ¹³C values.

A similar experiment was performed to examine whether two differentmethanotrophs, Methylococcus capsulatus Bath and Methylosinustrichosporium OB3b, grown on different methane sources and in variousbatches of bottle cultures showed a difference in δ¹³C distribution (seeTable 6).

TABLE 6 Stable Carbon Isotope Distribution of Different MethanotrophsGrown on Different Methane Sources of Different Purity Strain Methane*Batch No. Time (h)† OD₆₀₀ DCW (g/L) δ¹³C Cells Mc Bath A 62I 18 0.4940.18 −54.3 40 2.33 0.83 −42.1 48 3.08 1.09 −37.1 Mc Bath D 62J 18 0.5920.21 −38.3 40 1.93 0.69 −37.8 48 2.5 0.89 −37.8 Mc Bath D 62K 18 0.5640.20 −38.6 40 1.53 0.54 −37.5 48 2.19 0.78 −37.6 Mt OB3b A 68D 118 0.4220.24 −50.2 137 0.99 0.55 −47.7 162 1.43 0.80 −45.9 Mt OB3b A 68E 1180.474 0.26 −49.9 137 1.065 0.59 −47.6 162 1.51 0.84 −45.2 Mt OB3b D 68F118 0.534 0.30 −45.6 137 1.119 0.62 −38.7 162 1.63 0.91 −36.4 Mt OB3b D68G 118 0.544 0.30 −44.8 137 1.131 0.63 −39.1 162 1.6 0.89 −34.2*Methane sources and purity: A: 99% methane (grade 2.0); D: >93% methane(grade 1.3) †Time = bottle culture time in hours

The average δ¹³C for M capsulatus grown on a first methane source (A)was −44.5±8.8, while the average δ¹³C for M. trichosporium was −47.8±2.0grown on the same methane source. The average δ¹³C for M. capsulatusgrown on the second methane source (B) was −37.9±0.4, while the averageδ¹³C for M. trichosporium was −39.8±4.5. These data show that the δ¹³Cof cell material grown on a methane source is highly similar to the δ¹³Cof cell material from a different strain grown on the same source ofmethane. Thus, the observed δ¹³C of cell material appears to beprimarily dependent on the composition of the input gas rather than aproperty of a particular bacterial strain being studied.

The various embodiments described above can be combined to providefurther embodiments. All of the patent and non-patent publicationsreferred to in this specification or listed in the Application DataSheet, including the disclosure of U.S. provisional application No.61/928,366, filed Jan. 16, 2014, are incorporated herein by reference intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following embodiments,the terms used should not be construed to limit the embodiments to thespecific embodiments disclosed in the specification and the embodiments,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such embodiments are entitled.Accordingly, the embodiments are not limited by the disclosure.

What is claimed is:
 1. A recombinant methanotrophic bacterium,comprising an exogenous nucleic acid that encodes a gluconeogenesisenzyme, wherein the encoded gluconeogenesis enzyme is one or more of apyruvate carboxylase, a phosphoenolpyruvate carboxykinase, an enolase, aphosphoglycerate mutase, a phosphoglycerate kinase, aglyceraldehyde-3-phosphate dehydrogenase, a Type A aldolase, a fructose1,6-bisphosphatase, a phosphofructokinase, a phosphoglucose isomerase, ahexokinase, and a glucose-6-phosphate, wherein the methanotrophicbacterium is selected from the group consisting of Methylomonas,Methylobacter, Methylococcus, Methylosinus, Methylocystis,Methylomicrobium, Methanomonas, and Methylocella, and wherein therecombinant methanotrophic bacterium grown on a C₁ substrate feedstockis capable of producing glucose at a level that is greater than thatproduced by the parent methanotrophic bacterium.
 2. The recombinantmethanotrophic bacterium of claim 1, wherein the C₁ substrate feedstockcomprises natural gas or methane.
 3. The recombinant methanotrophicbacterium of claim 1, wherein the methanotrophic bacterium is anobligate methanotrophic bacterium.
 4. The recombinant methanotrophicbacterium of claim 1, wherein the methanotrophic bacterium is afacultative methanotrophic bacterium.
 5. The recombinant methanotrophicbacterium of claim 1, wherein the methanotrophic bacterium is selectedfrom the group consisting of Methylococcus capsulatus Bath, Methylomonassp. 16a, Methylosinus trichosporium OB3b, Methylosinus sporium,Methylocystis parvus, Methylomonas methanica, Methylomonas albus,Methylobacter capsulatus Y, Methylomonas flagellata AJ-3670,Methylacidiphilum infernorum, Methylacidiphdum fumariolicum,Methylomicrobium alcaliphdum, and Methyloacida kamchatkensis.
 6. Therecombinant methanotrophic bacterium of claim 1, wherein the exogenousnucleic acid encoding the gluconeogenesis enzyme is endogenous to anorganism selected from the group consisting of a bacterium, a yeast, afungi, and a plant.
 7. The recombinant methanotrophic bacterium of claim1, wherein the exogenous nucleic acid comprises an expression controlsequence that is operably linked to the nucleic acid encoding thegluconeogenesis enzyme.
 8. The recombinant methanotrophic bacterium ofclaim 1, wherein the exogenous nucleic acid encoding the gluconeogenesisenzyme is endogenous to Escherichia coli or Corynebacterium glutamicum.9. The recombinant methanotrophic bacterium of claim 1, wherein theexogenous nucleic acid encoding the gluconeogenesis enzyme is endogenousto Saccharomyces cerevisiae.
 10. The recombinant methanotrophicbacterium of claim 1, wherein the exogenous nucleic acid encoding thegluconeogenesis enzyme is codon optimized for the methanotrophicbacterium.
 11. The recombinant methanotrophic bacterium of claim 1,wherein the carbohydrates of the recombinant methanotrophic bacteriumexhibits a δ¹³C that is less than −30‰ or is less than −40‰.
 12. Therecombinant methanotrophic bacterium of claim 1, wherein themethanotrophic bacterium further comprises an exogenous polynucleotideencoding one or more glycogenesis enzymes selected from the groupconsisting of a glucose-1-phosphate adenyltransferase, a glycogensynthase, and a 1,4-alpha-glucan-branching protein.
 13. The recombinantmethanotrophic bacterium of claim 12, wherein the further exogenouspolynucleotide encoding one or more glycogenesis enzymes comprises anexpression control sequence that is operably linked to thepolynucleotide encoding the one or more glycogenesis enzymes, whereinthe one or more glycogenesis enzymes are heterologous glycogenesisenzymes, native glycogenesis enzymes, or a combination thereof.
 14. Therecombinant methanotrophic bacterium of claim 12, wherein the exogenousnucleic acid encoding the one or more glycogenesis enzymes is codonoptimized for the methanotrophic bacterium.
 15. A biomass derived fromwhole and/or lysed cells of the recombinant methanotrophic bacterium ofclaim
 1. 16. A carbohydrate composition, comprising carbohydratesextracted from a biomass derived from the methanotrophic bacterium ofclaim 1, wherein the composition exhibits a δ¹³C that is less than −30‰or is less than −40‰.
 17. An animal feed, comprising the recombinantmethanotrophic bacterium of claim 1, a biomass derived from whole and/orlysed cells of the recombinant methanotrophic bacterium of claim 1, or acarbohydrate composition comprising carbohydrates extracted from abiomass derived from the recombinant methanotrophic bacterium ofclaim
 1. 18. The animal feed of claim 17, further comprising an additiveselected from the group consisting of a plant-derived material, ananimal-derived material, and a microorganism-derived material.
 19. Theanimal feed of claim 18, wherein the additive is plant-derived materialand the plant-derived material is selected from the group consisting ofcorn, soybean meal, pea protein, or a combination thereof.
 20. Theanimal feed of claim 18, wherein the additive is an animal-derivedmaterial and the animal-derived material is fish meal.
 21. A method ofproducing a desired carbohydrate, the method comprising culturing therecombinant methanotrophic bacterium of claim 1 in the presence of a C₁substrate feedstock comprising methane under conditions sufficient toproduce the glucose.
 22. The method of claim 21, wherein the naturalgas-derived carbon feedstock is natural gas.
 23. The method of claim 21,wherein the methanotrophic bacterium is selected from the groupconsisting of Methylococcus capsulatus Bath, Methylomonas sp. 16a,Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystisparvus, Methylomonas methanica, Methylomonas albus, Methylobactercapsulatus Y, Methylomonas flagellata AJ-3670, Methylacidiphiluminfernorum, Methylacidiphilum fumariolicum, Methylomicrobiumalcaliphilum, and Methyloacida kamchatkensis.